Evolved sortases and uses thereof
10202593 ยท 2019-02-12
Assignee
Inventors
Cpc classification
C12P21/02
CHEMISTRY; METALLURGY
International classification
C12P21/02
CHEMISTRY; METALLURGY
C12N9/00
CHEMISTRY; METALLURGY
Abstract
Evolved sortases exhibiting enhanced reaction kinetics and/or altered substrate preferences are provided herein, for example evolved sortases that bind recognitions motifs comprising a LAXT or LPXS sequence. Also provided are methods (e.g., orthogonal transpeptidation and diagnostics methods) for using such sortases. Kits comprising materials, reagents, and cells for carrying out the methods described herein are also provided.
Claims
1. A Staphylococcus aureus (S. aureus) Sortase A variant that binds substrates comprising the sequence LAXT, wherein X represents any amino acid, and wherein the S. aureus Sortase A variant comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of S. aureus Sortase A as provided as SEQ ID NO: 1, and wherein the amino acid sequence of the Sortase A variant includes a mutation selected from the group consisting of K84R, R99H, R99K, S102C, A104H, E105D, K138I, K138V, K138P, K145E, K152I, D160K, K162R, K162H, T164N, V168I, K177G, K177R, I182F, and K196S.
2. The Staphylococcus aureus (S. aureus) Sortase A variant of claim 1, wherein the S. aureus Sortase A variant comprises an amino acid sequence that is at least 95% identical to the amino acid sequence provided in SEQ ID NO: 1.
3. The Staphylococcus aureus (S. aureus) Sortase A variant of claim 1, wherein the amino acid sequence of the S. aureus Sortase A variant comprises at least two mutations, as compared to the amino acid sequence of S. aureus Sortase A provided as SEQ ID NO: 1.
4. The Staphylococcus aureus (S. aureus) Sortase A variant of claim 1, wherein the substrate comprises the amino acid sequence LAXTX, wherein each occurrence of X independently represents any amino acid.
5. The Staphylococcus aureus (S. aureus) Sortase A variant of claim 1, wherein the substrate comprises the amino acid sequence LAETG (SEQ ID NO: 5).
6. A Staphylococcus aureus (S. aureus) Sortase A variant that binds substrates comprising the sequence LPXS, wherein X represents any amino acid, the S. aureus Sortase A variant comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of S. aureus Sortase A as provided as SEQ ID NO: 1, wherein the amino acid sequence of the Sortase A variant includes a mutation selected from the group consisting of N98D, S102C, A104V, A118S, A118T, F122A, K134G, K134P, E189V, E189F, and E189P.
7. A method for transpeptidation, the method comprising contacting the S. aureus Sortase A variant of claim 1 with a substrate comprising an LAXT amino acid sequence, wherein X represents any amino acid, and a substrate comprising a GGG sequence under conditions suitable for sortase-mediated transpeptidation.
8. A method for transpeptidation comprising contacting the Staphylococcus aureus (S. aureus) Sortase A variant of claim 6 with a substrate comprising an LPXS amino acid sequence, wherein X represents any amino acid, and a substrate comprising a GGG sequence under conditions suitable for sortase-mediated transpeptidation.
9. A method for N-terminal protein modification comprising contacting a protein comprising a N-terminal GGG sequence with the Staphylococcus aureus (S. aureus) Sortase A variant of claim 1, and a sortase substrate comprising a LAXT sequence, respectively, under conditions suitable for sortase-mediated transpeptidation, wherein X represents any amino acid.
10. A method for C-terminal protein modification comprising contacting a protein comprising a C-terminal LAXT sequence with the Staphylococcus aureus (S. aureus) Sortase A variant of claim 1, respectively, and a sortase substrate comprising a GGG sequence under conditions suitable for sortase-mediated transpeptidation, wherein X represents any amino acid.
11. A method for modifying a protein comprising a sortase recognition motif in a cell or tissue, the method comprising contacting the protein with the Staphylococcus aureus (S. aureus) Sortase A variant of claim 1 and a sortase substrate comprising a sortase recognition motif under conditions suitable for sortase-mediated transpeptidation, wherein (a) the protein comprises a N-terminal sortase recognition motif, and the sortase substrate comprises a C-terminal sortase recognition motif; or (b) the protein comprises a C-terminal sortase recognition motif, and the sortase substrate comprises a N-terminal sortase recognition motif, wherein the N-terminal sortase recognition motif comprises the sequence GGG, and the C-terminal sortase recognition motif comprises the sequence LAXT, wherein X represents any amino acid.
12. The Staphylococcus aureus (S. aureus) sortase A variant of claim 1, wherein the amino acid sequence of the Sortase A variant includes two mutations selected from the group consisting of K84R, R99H, R99K, S102C, A104H, E105D, K138I, K138V, K138P, K145E, K152I, D160K, K162R, K162H, T164N, V168I, K177G, K177R, I182F, and K196S.
13. The Staphylococcus aureus (S. aureus) sortase A variant of claim 1, wherein the amino acid sequence of the Sortase A variant includes three mutations selected from the group consisting of K84R, R99H, R99K, S102C, A104H, E105D, K138I, K138V, K138P, K145E, K152I, D160K, K162R, K162H, T164N, V168I, K177G, K177R, I182F, and K196S.
14. The Staphylococcus aureus (S. aureus) sortase A variant of claim 1, wherein the amino acid sequence of the Sortase A variant includes four mutations selected from the group consisting of K84R, R99H, R99K, S102C, A104H, E105D, K138I, K138V, K138P, K145E, K152I, D160K, K162R, K162H, T164N, V168I, K177G, K177R, I182F, and K196S.
15. The Staphylococcus aureus (S. aureus) Sortase A variant of claim 1, wherein the S. aureus Sortase A variant comprises an amino acid sequence that is at least 98% identical to the amino acid sequence provided in SEQ ID NO: 1.
16. The Staphylococcus aureus (S. aureus) Sortase A variant of claim 1, wherein the S. aureus Sortase A variant exhibits a ratio of k.sub.cat/K.sub.M for a substrate comprising the amino acid sequence LAETG (SEQ ID NO: 5) that is least 60-fold greater than the K.sub.cat/K.sub.M ratio the sortase exhibits for a substrate comprising the amino acid sequence LPETG (SEQ ID NO: 4).
17. The Staphylococcus aureus (S. aureus) Sortase A variant of claim 1, wherein the S. aureus Sortase A variant exhibits a ratio of k.sub.cat/K.sub.M for a substrate comprising the amino acid sequence LAETG (SEQ ID NO: 5) that is least 100-fold greater than the K.sub.cat/K.sub.M ratio the sortase exhibits for a substrate comprising the amino acid sequence LPETG (SEQ ID NO: 4).
18. The Staphylococcus aureus (S. aureus) Sortase A variant of claim 1, wherein the S. aureus Sortase A variant exhibits a ratio of k.sub.cat/K.sub.M for a substrate comprising the amino acid sequence LAETG (SEQ ID NO: 5) that is least 140-fold greater than the K.sub.cat/K.sub.M ratio the sortase exhibits for a substrate comprising the amino acid sequence LPETG (SEQ ID NO: 4).
19. The Staphylococcus aureus (S. aureus) Sortase A variant of claim 1, wherein the S. aureus Sortase A variant exhibits a K.sub.M for a substrate comprising the amino acid sequence LAETG (SEQ ID NO: 5) that is at least 15-fold less than the K.sub.M for substrates comprising the amino acid sequence LPETG (SEQ ID NO: 4).
20. The Staphylococcus aureus (S. aureus) Sortase A variant of claim 1, wherein the S. aureus Sortase A variant exhibits a K.sub.M for a substrate comprising the amino acid sequence LAETG (SEQ ID NO: 5) that is at least 20-fold less than the K.sub.M for substrates comprising the amino acid sequence LPETG (SEQ ID NO: 4).
21. The Staphylococcus aureus (S. aureus) sortase A variant of claim 6, wherein the amino acid sequence of the Sortase A variant includes two mutations selected from the group consisting of N98D, S102C, A104V, A118S, A118T, F122A, K134G, K134P, E189V, E189F, and E189P.
22. The Staphylococcus aureus (S. aureus) sortase A variant of claim 6, wherein the amino acid sequence of the Sortase A variant includes three mutations selected from the group consisting of N98D, S102C, A104V, A118S, A118T, F122A, K134G, K134P, E189V, E189F, and E189P.
23. The Staphylococcus aureus (S. aureus) sortase A variant of claim 6, wherein the amino acid sequence of the Sortase A variant includes four mutations selected from the group consisting of N98D, S102C, A104V, A118S, A118T, F122A, K134G, K134P, E189V, E189F, and E189P.
24. The Staphylococcus aureus (S. aureus) Sortase A variant of claim 6, wherein the S. aureus Sortase A variant comprises an amino acid sequence that is at least 95% identical to the amino acid sequence provided in SEQ ID NO: 1.
25. The Staphylococcus aureus (S. aureus) Sortase A variant of claim 6, wherein the S. aureus Sortase A variant comprises an amino acid sequence that is at least 98% identical to the amino acid sequence provided in SEQ ID NO: 1.
26. A method for N-terminal protein modification comprising contacting a protein comprising a N-terminal GGG sequence with a the Staphylococcus aureus (S. aureus) Sortase A variant of claim 6, and a sortase substrate comprising a LPXS sequence, respectively, under conditions suitable for sortase-mediated transpeptidation, wherein X represents any amino acid.
27. A method for C-terminal protein modification comprising contacting a protein comprising a C-terminal LPXS sequence with a the Staphylococcus aureus (S. aureus) Sortase A variant of claim 6, respectively, and a sortase substrate comprising a GGG sequence under conditions suitable for sortase-mediated transpeptidation, wherein X represents any amino acid.
28. A method for modifying a protein comprising a sortase recognition motif in a cell or tissue, the method comprising contacting the protein with the Staphylococcus aureus (S. aureus) Sortase A variant of claim 6 and a sortase substrate comprising a sortase recognition motif under conditions suitable for sortase-mediated transpeptidation, wherein (a) the protein comprises a N-terminal sortase recognition motif, and the sortase substrate comprises a C-terminal sortase recognition motif; or (b) the protein comprises a C-terminal sortase recognition motif, and the sortase substrate comprises a N-terminal sortase recognition motif, wherein the N-terminal sortase recognition motif comprises the sequence GGG, and the C-terminal sortase recognition motif comprises the sequence LPXS, wherein X represents any amino acid.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DEFINITIONS
(27) The term agent, as used herein, refers to any molecule, entity, or moiety. For example, an agent may be a protein, an amino acid, a peptide, a polynucleotide, a carbohydrate, a lipid, a detectable label, a binding agent, a tag, a metal atom, a contrast agent, a catalyst, a non-polypeptide polymer, a synthetic polymer, a recognition element, a linker, or chemical compound, such as a small molecule. In some embodiments, the agent is a binding agent, for example, a ligand, a ligand-binding molecule, an antibody, or an antibody fragment. In some embodiments, the term modifying agent is used interchangeably with agent. Additional agents suitable for use in embodiments of the present invention will be apparent to the skilled artisan. The invention is not limited in this respect.
(28) The term amino acid, as used herein, includes any naturally occurring and non-naturally occurring amino acid. Suitable natural and non-natural amino acids will be apparent to the skilled artisan, and include, but are not limited to, those described in S. Hunt, The Non-Protein Amino Acids: In Chemistry and Biochemistry of the Amino Acids, edited by G. C. Barrett, Chapman and Hall, 1985. Some non-limiting examples of non-natural amino acids are 4-hydroxyproline, desmosine, gamma-aminobutyric acid, beta-cyanoalanine, norvaline, 4-(E)-butenyl-4(R)-methyl-N-methyl-L-threonine, N-methyl-L-leucine, 1-amino-cyclopropanecarboxylic acid, 1-amino-2-phenyl-cyclopropanecarboxylic acid, 1-amino-cyclobutanecarboxylic acid, 4-amino-cyclopentenecarboxylic acid, 3-amino-cyclohexanecarboxylic acid, 4-piperidylacetic acid, 4-amino-1-methylpyrrole-2-carboxylic acid, 2,4-diaminobutyric acid, 2,3-diaminopropionic acid, 2,4-diaminobutyric acid, 2-aminoheptanedioic acid, 4-(aminomethyl)benzoic acid, 4-aminobenzoic acid, ortho-, meta- and para-substituted phenylalanines (e.g., substituted with C(O)C.sub.6H.sub.5; CF.sub.3; CN; -halo; NO.sub.2; CH.sub.3), disubstituted phenylalanines, substituted tyrosines (e.g., further substituted with C(O)C.sub.6H.sub.5; CF.sub.3; CN; -halo; NO.sub.2; CH.sub.3), and statine. In the context of amino acid sequences, X or Xaa represents any amino acid residue, e.g., any naturally occurring and/or any non-naturally occurring amino acid residue.
(29) The term antibody, as used herein, refers to a protein belonging to the immunoglobulin superfamily. The terms antibody and immunoglobulin are used interchangeably. Antibodies from any mammalian species (e.g., human, mouse, rat, goat, pig, horse, cattle, camel) and from non-mammalian species (e.g., from non-mammalian vertebrates, birds, reptiles, amphibia) are within the scope of the term. Suitable antibodies and antibody fragments for use in the context of some embodiments of the present invention include, for example, human antibodies, humanized antibodies, domain antibodies, F(ab), F(ab)2, Fab, Fv, Fc, and Fd fragments, antibodies in which the Fc and/or FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; antibodies in which the FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; antibodies in which the FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; and antibodies in which the FR and/or CDR1 and/or CDR2 regions have been replaced by homologous human or non-human sequences. In some embodiments, so-called single chain antibodies (e.g., ScFv), (single) domain antibodies, and other intracellular antibodies may be used in the context of the present invention. Domain antibodies, camelid and camelized antibodies and fragments thereof, for example, VHH domains, or nanobodies, such as those described in patents and published patent applications of Ablynx NV and Domantis are also encompassed in the term antibody. Further, chimeric antibodies, e.g., antibodies comprising two antigen-binding domains that bind to different antigens, are also suitable for use in the context of some embodiments of the present invention.
(30) The term binding agent, as used herein refers to any molecule that binds another molecule. In some embodiments, a binding agent binds another molecule with high affinity. In some embodiments, a binding agent binds another molecule with high specificity. The binding agent may be a protein, peptide, nucleic acid, carbohydrate, polymer, or small molecule. Examples for binding agents include, without limitation, antibodies, antibody fragments, receptors, ligands, aptamers, receptors, and adnectins.
(31) The term bond-forming enzyme, as used herein, refers to any enzyme that catalyzes a reaction resulting in the formation of a covalent bond. In some embodiments, the bond-forming enzyme is a sortase.
(32) The term conjugated or conjugation refers to an association of two entities, for example, of two molecules such as two proteins, or a protein and a reactive handle, or a protein and an agent, e.g., a detectable label. The association can be, for example, via a direct or indirect (e.g., via a linker) covalent linkage or via non-covalent interactions. In some embodiments, the association is covalent. In some embodiments, two molecules are conjugated via a linker connecting both molecules. For example, in some embodiments where two proteins are conjugated to each other to form a protein fusion, the two proteins may be conjugated via a polypeptide linker, e.g., an amino acid sequence connecting the C-terminus of one protein to the N-terminus of the other protein. In some embodiments, conjugation of a protein to a protein or peptide is achieved by transpeptidation using a sortase. See, e.g., Ploegh et al., International PCT Patent Application, PCT/US2010/000274, filed Feb. 1, 2010, published as WO/2010/087994 on Aug. 5, 2010, Ploegh et al., International Patent Application PCT/US2011/033303, filed Apr. 20, 2011, published as WO/2011/133704 on Oct. 27, 2011, Chaikof et al., U.S. Provisional Patent Application Ser. No. 61/720,294, filed Oct. 30, 2012, and Liu et al., U.S. patent application Ser. No. 13/922,812, filed Jun. 20, 2013 the entire contents of each of which are incorporated herein by reference, for exemplary sortases, proteins, recognition motifs, reagents, and methods for sortase-mediated transpeptidation.
(33) The term detectable label refers to a moiety that has at least one element, isotope, or functional group incorporated into the moiety which enables detection of the molecule, e.g., a protein or peptide, or other entity, to which the label is attached. Labels can be directly attached or can be attached via a linker. It will be appreciated that the label may be attached to or incorporated into a molecule, for example, a protein, polypeptide, or other entity, at any position. In general, a detectable label can fall into any one (or more) of five classes: I) a label which contains isotopic moieties, which may be radioactive or heavy isotopes, including, but not limited to, .sup.2H, .sup.3H, .sup.13C, .sup.17C, .sup.15N, .sup.18F, .sup.31P, .sup.32P, .sup.35S, .sup.67Ga, .sup.76Br, .sup.99mTc (Tc-.sup.99m), .sup.111In, .sup.125I, .sup.131I, .sup.153Gd, .sup.169Yb, and .sup.186Re; II) a label which contains an immune moiety, which may be antibodies or antigens, which may be bound to enzymes (e.g., such as horseradish peroxidase); III) a label which is a colored, luminescent, phosphorescent, or fluorescent moieties (e.g., such as the fluorescent label fluorescein-isothiocyanate (FITC); IV) a label which has one or more photo affinity moieties; and V) a label which is a ligand for one or more known binding partners (e.g., biotin-streptavidin, FK506-FKBP). In certain embodiments, a label comprises a radioactive isotope, preferably an isotope which emits detectable particles, such as particles. In certain embodiments, the label comprises a fluorescent moiety. In certain embodiments, the label is the fluorescent label fluorescein-isothiocyanate (FITC). In certain embodiments, the label comprises a ligand moiety with one or more known binding partners. In certain embodiments, the label comprises biotin, which may be detected using a streptavidin conjugate (e.g., fluorescent streptavidin conjugates such as Streptavidin ALEXA FLUOR 568 conjugate (SA-568) and Streptavidin ALEXA FLUOR 800 conjugate (SA-800), Invitrogen). In some embodiments, a label is a fluorescent polypeptide (e.g., GFP or a derivative thereof such as enhanced GFP (EGFP)) or a luciferase (e.g., a firefly, Renilla, or Gaussia luciferase). It will be appreciated that, in certain embodiments, a label may react with a suitable substrate (e.g., a luciferin) to generate a detectable signal. Non-limiting examples of fluorescent proteins include GFP and derivatives thereof, proteins comprising fluorophores that emit light of different colors such as red, yellow, and cyan fluorescent proteins. Exemplary fluorescent proteins include, e.g., Sirius, Azurite, EBFP2, TagBFP, mTurquoise, ECFP, Cerulean, TagCFP, mTFP1, mUkG1, mAG1, AcGFP1, TagGFP2, EGFP, mWasabi, EmGFP, TagYPF, EYFP, Topaz, SYFP2, Venus, Citrine, mKO, mKO2, mOrange, mOrange2, TagRFP, TagRFP-T, mStrawberry, mRuby, mCherry, mRaspberry, mKate2, mPlum, mNeptune, T-Sapphire, mAmetrine, mKeima. See, e.g., Chalfie, M. and Kain, S R (eds.) Green fluorescent protein: properties, applications, and protocols Methods of biochemical analysis, v. 47 Wiley-Interscience, Hoboken, N.J., 2006; and Chudakov, D M, et al., Physiol Rev. 90(3):1103-63, 2010, for discussion of GFP and numerous other fluorescent or luminescent proteins. In some embodiments, a label comprises a dark quencher, e.g., a substance that absorbs excitation energy from a fluorophore and dissipates the energy as heat.
(34) The term homologous, as used herein is an art understood term that refers to nucleic acids or polypeptides that are highly related at the level of nucleotide or amino acid sequence. Nucleic acids or polypeptides that are homologous to each other are termed homologues. Homology between two sequences can be determined by sequence alignment methods known to those of skill in the art. For example, the homology, or percent identity of two amino acid sequences can be determined using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, modified as in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. J. Mol. Biol. 215:403-10, 1990. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to the protein molecules of interest. Where gaps exist between two sequences, Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. In accordance with the invention, two sequences are considered to be homologous if they are at least about 50-60% identical, e.g., share identical residues (e.g., amino acid residues) in at least about 50-60% of all residues comprised in one or the other sequence, at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical, for at least one stretch of at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 120, at least 150, or at least 200 amino acids.
(35) The term k.sub.cat refers to the turnover rate of an enzyme, e.g., the number of substrate molecules that the respective enzyme converts to product per time unit. Typically, k.sub.cat designates the turnover of an enzyme working at maximum efficiency.
(36) The term K.sub.M is used herein interchangeably with the term K.sub.m and refers to the Michaelis constant of an enzyme, an art-recognized measure designating the substrate concentration at the maximum reaction velocity of a reaction catalyzed by the respective enzyme.
(37) The term linker, as used herein, refers to a chemical group or molecule covalently linked to a molecule, for example, a protein, and a chemical group or moiety, for example, a click chemistry handle. In some embodiments, the linker is positioned between, or flanked by, two groups, molecules, or other moieties and connected to each one via a covalent bond, thus connecting the two. In some embodiments, the linker is an amino acid or a plurality of amino acids (e.g., a peptide or protein). In some embodiments, the linker is an organic molecule, group, polymer (e.g., PEG), or chemical moiety.
(38) The term mutation, as used herein, refers to a substitution of a residue within a sequence, e.g., a nucleic acid or amino acid sequence, with another residue, or a deletion or insertion of one or more residues within a sequence. Mutations are typically described herein by identifying the original residue followed by the position of the residue within the sequence and by the identity of the newly substituted residue. For example, the term P94S in the context of describing a mutation in the S. aureus sortase A protein describes a mutation in which the P (proline) residue at position 94 in the sortase A sequence has been replaced by an S (serine) residue, the term P94R describes a mutation in which the P (proline) residue at position 94 in the sortase A sequence has been replaced by an R (arginine) residue, the term E106G describes a mutation in which the E (glutamate) residue at position 106 in the sortase A sequence has been replaced by a G (glycine) residue, and so forth. See, e.g., SEQ ID NO: 1 for reference of the respective amino acid residue positions in the wild type S. aureus sortase A protein. It should be appreciated that methods for making the amino acid substitutions (mutations) provided herein are well known in the art, and are provided by, for example, Green and Sambrook, Molecular Cloning: A Laboratory Manual (4.sup.th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)).
(39) The terms protein, peptide, and polypeptide are used interchangeably herein, and refer to a polymer of amino acid residues linked together by peptide (amide) bonds. The terms refer to a protein, peptide, or polypeptide of any size, structure, or function. Typically, a protein, peptide, or polypeptide will be at least three amino acids long. A protein, peptide, or polypeptide may refer to an individual protein or a collection of proteins. One or more of the amino acids in a protein, peptide, or polypeptide may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a hydroxyl group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation, functionalization, or other modification, etc. A protein, peptide, or polypeptide may also be a single molecule or may be a multi-molecular complex. A protein, peptide, or polypeptide may be just a fragment of a naturally occurring protein or peptide. A protein, peptide, or polypeptide may be naturally occurring, recombinant, or synthetic, or any combination thereof.
(40) The term reactive handle, as used herein, refers to a reactive moiety that can partake in a bond-forming reaction under physiological conditions. Reactive handles can be used to conjugate entities with reactive handles that can react with each other. Examples of suitable reactive handles are, for example, chemical moieties that can partake in a click chemistry reaction (see, e.g., H. C. Kolb, M. G. Finn and K. B. Sharpless (2001). Click Chemistry: Diverse Chemical Function from a Few Good Reactions. Angewandte Chemie International Edition 40 (11): 2004-2021). Some suitable reactive handles are described herein and additional suitable reactive handles will be apparent to those of skill in this art, as the present invention is not limited in this respect.
(41) The term small molecule is used herein to refer to molecules, whether naturally-occurring or artificially created (e.g., via chemical synthesis) that have a relatively low molecular weight. Typically, a small molecule is an organic compound (i.e., it contains carbon). A small molecule may contain multiple carbon-carbon bonds, stereocenters, and other functional groups (e.g., amines, hydroxyl, carbonyls, or heterocyclic rings). In some embodiments, small molecules are monomeric and have a molecular weight of less than about 1500 g/mol. In certain embodiments, the molecular weight of the small molecule is less than about 1000 g/mol or less than about 500 g/mol. In certain embodiments, the small molecule is a drug, for example, a drug that has already been deemed safe and effective for use in humans or animals by the appropriate governmental agency or regulatory body.
(42) The term sortase, as used herein, refers to a protein having sortase activity, i.e., an enzyme able to carry out a transpeptidation reaction conjugating the C-terminus of a protein (or the C-terminus of a peptide conjugate, i.e., an agent comprising a peptide) to the N-terminus of a protein (or the N-terminus of a peptide conjugate, i.e., an agent comprising a peptide) via transamidation. The term includes full-length sortase proteins, e.g., full-length naturally occurring sortase proteins, fragments of such sortase proteins that have sortase activity, modified (e.g., mutated) variants or derivatives of such sortase proteins or fragments thereof, as well as proteins that are not derived from a naturally occurring sortase protein, but exhibit sortase activity. Those of skill in the art will readily be able to determine whether or not a given protein or protein fragment exhibits sortase activity, e.g., by contacting the protein or protein fragment in question with a suitable sortase substrate under conditions allowing transpeptidation and determining whether the respective transpeptidation reaction product is formed. In some embodiments, a sortase is a protein comprising at least 20 amino acid residues, at least 30 amino acid residues, at least 40 amino acid residues, at least 50 amino acid residues, at least 60 amino acid residues, at least 70 amino acid residues, at least 80 amino acid residues, at least 90 amino acid residues, at least 100 amino acid residues, at least 125 amino acid residues, at least 150 amino acid residues, at least 175 amino acid residues, at least 200 amino acid residues, or at least 250 amino acid residues. In some embodiments, a sortase is a protein comprising less than 100 amino acid residues, less than 125 amino acid residues, less than 150 amino acid residues, less than 175 amino acid residues, less than 200 amino acid residues, or less than 250 amino acid residues. In some embodiments, the sortase comprises a sortase catalytic domain and, optionally, an additional domain, e.g., a transmembrane domain.
(43) Suitable sortases will be apparent to those of skill in the art and include, but are not limited to, sortase A, sortase B, sortase C, and sortase D type sortases. Suitable sortases are described, for example, in Dramsi S, Trieu-Cuot P, Bierne H, Sorting sortases: a nomenclature proposal for the various sortases of Gram-positive bacteria. Res Microbiol. 156(3):289-97, 2005; Comfort D, Clubb R T. A comparative genome analysis identifies distinct sorting pathways in gram-positive bacteria. Infect Immun., 72(5):2710-22, 2004; Chen I, Don B M, and Liu D R., A general strategy for the evolution of bond-forming enzymes using yeast display. Proc Natl Acad Sci USA. 2011 Jul. 12; 108(28):11399; and Pallen, M. J.; Lam, A. C.; Antonio, M.; Dunbar, K. TRENDS in Microbiology, 2001, 9(3), 97-101; the entire contents of each of which are incorporated herein by reference). Amino acid sequences of sortases and the nucleotide sequences that encode them are known to those of skill in the art and are disclosed in a number of references cited herein, the entire contents of all of which are incorporated herein by reference. Those of skill in the art will appreciate that any sortase and any sortase recognition motif can be used in some embodiments of this invention, including, but not limited to, the sortases and sortase recognition motifs described in Ploegh et al., International PCT Patent Application, PCT/US2010/000274, filed Feb. 1, 2010, published as WO/2010/087994 on Aug. 5, 2010; and Ploegh et al., International Patent Application PCT/US2011/033303, filed Apr. 20, 2011, published as WO/2011/133704 on Oct. 27, 2011; the entire contents of each of which are incorporated herein by reference.
(44) In some embodiments, the sortase is sortase A of S. aureus. For example, in some embodiments, wild type sortase A from S. aureus serves as the starting sortase, or parent sortase, for generating the sortase mutants and their methods of use disclosed herein. The amino acid sequence of wild type sortase A of S. aureus is known to those of skill in the art, and a representative sequence (gi|21284177|ref|NP_647265.1) is provided below:
(45) TABLE-US-00001 (SEQIDNO:1) MKKWTNRLMTIAGVVLILVAAYLFAKPHIDNYLHDKDKDEKIEQYD KNVKEQASKDKKQQAKPQIPKDKSKVAGYIEIPDADIKEPVYPGPA TPEQLNRGVSFAEENESLDDQNISIAGHTFIDRPNYQFTNLKAAKK GSMVYFKVGNETRKYKMTSIRDVKPTDVEVLDEQKGKDKQLTLITC DDYNEKTGVWEKRKIFVATEVK.
Additional S. aureus sortase A sequences will be apparent to those of skill in the art, and the invention is not limited in this respect. In some embodiments, the sortase is a sortase A of another organism, for example, from another bacterial strain, such as S. pyogenes. In some embodiments, the sortase is a sortase B, a sortase C, or a sortase D. Suitable sortases from other bacterial strains will be apparent to those of skill in the art, and the invention is not limited in this respect.
(46) The term sortase substrate, as used herein refers to a molecule or entity that can be utilized in a sortase-mediated transpeptidation reaction. Typically, a sortase utilizes two substratesa substrate comprising a C-terminal sortase recognition motif, and a second substrate comprising an N-terminal sortase recognition motif and the transpeptidation reaction results in a conjugation of both substrates via a covalent bond. In some embodiments the C-terminal and N-terminal recognition motif are comprised in the same protein, e.g., in the same amino acid sequence. Sortase-mediated conjugation of the substrates in such cases results in the formation of an intramolecular bond, e.g., a circularization of a single amino acid sequence, or, if multiple polypeptides of a protein complex are involved, the formation of an intra-complex bond. In some embodiments, the C-terminal and N-terminal recognition motifs are comprised in different amino acid sequences, for example, in separate proteins or other agents. Some sortase recognition motifs are described herein and additional suitable sortase recognition motifs are well known to those of skill in the art. For example, sortase A of S. aureus recognizes and utilizes a C-terminal LPXT motif and an N-terminal GGG motif in transpeptidation reactions. In some embodiments, the LPXT motif comprises a C-terminal glycine (e.g., LPXTG; SEQ ID NO: 2). Additional sortase recognition motifs will be apparent to those of skill in the art, and the invention is not limited in this respect. A sortase substrate may comprise additional moieties or entities apart from the peptidic sortase recognition motif. For example, a sortase substrate may comprise an LPXT motif, the N-terminus of which is conjugated to any agent, e.g., a peptide or protein, a small molecule, a binding agent, a lipid, a carbohydrate, or a detectable label. Similarly, a sortase substrate may comprise a GGG motif, the C-terminus of which is conjugated to any agent, e.g., a peptide or protein, a small molecule, a binding agent, a lipid, a carbohydrate, or a detectable label. Accordingly, sortase substrates are not limited to proteins or peptides but include any moiety or entity conjugated to a sortase recognition motif.
(47) The term target protein, as used herein refers to a protein that comprises a sortase recognition motif. A target protein may be a wild type protein, or may be an engineered protein, e.g., a recombinant protein.
DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION
(48) The extent and diversity of applications utilizing sortases as catalysts for transpeptidation reactions remain limited by the difficulty of finding in nature or creating in the laboratory highly active sortases that bind substrates having recognition motifs other than the canonical, or wild type motif. One method for creating such sortases in the laboratory is through directed evolution.
(49) Accordingly, some embodiments of this invention provide novel evolved sortases generated by a directed evolution technology based on an integration of cell display (e.g., yeast display), enzyme-catalyzed small molecule-protein conjugation, and FACS, which provides a general strategy for the evolution of proteins that catalyze bond-forming reactions. See, e.g., Liu et al., U.S. patent application Ser. No. 13/922,812, filed Jun. 20, 2013; Chen I, Don B M, and Liu D R., A general strategy for the evolution of bond-forming enzymes using yeast display. Proc Natl Acad Sci USA. 2011 Jul. 12; 108(28):11399, the entire contents of each are incorporated herein by reference, for exemplary methods for evolving sortases. The technology was previously applied to evolve the bacterial transpeptidase sortase A of Staphylococcus aureus for improved catalytic activity, resulting in sortase variants with an improvement in activity of up to 140-fold. See, e.g., Liu et al., U.S. patent application Ser. No. 13/922,812, filed Jun. 20, 2013. As provided herein, the technology was applied to evolve sortase A of S. aureus for improved catalytic activity and altered substrate specificity, resulting in sortase variants with an improvement in activity and/or efficiency (e.g., of up to 850-fold increase in k.sub.cat/K.sub.m) for substrates with altered recognition motifs.
(50) Other aspects of this invention relate to kits including the evolved sortases described herein and methods of using such sortases, for example, in orthogonal protein modification (e.g., modifying a protein at its N-terminus, C-terminus, or both), as well as cell and tissue modification (e.g., modifying proteins within or on a cell or tissue). In one example, evolved sortases and methods are provided which allow for the modification, either in vivo or in vitro, of proteins at both the N- and C-termini, for example using two different sortases (e.g., from those described herein) to catalyze transpeptidation reactions at each end of a protein. Such modifications can, for example, alter the function of the protein, provide a means of detecting the protein, alter its bioavailability and/or half-life (e.g., in the context of a protein therapeutic), or combinations thereof. In another example, the sortases and methods provided herein allow for cell and tissue modification, for example by conjugating agents to the surface of a cell or tissue. Such modifications are useful, for example, in therapeutic contexts, such as cell or tissue transplantation (e.g., by conjugating anti-clotting factors, and/or anti-bacterial agents).
(51) Sortases
(52) This invention provides evolved sortases that efficiently use substrates not typically used by the respective parent wild-type sortase, e.g., substrates comprising the amino acid sequence LAXT or LPXS. For example, in some embodiments, an evolved sortase is provided that is derived from a wild type S. aureus sortase A as the parent sortase, which utilizes substrates comprising a C-terminal sortase recognition motif (e.g., an LPXT motif) and substrates comprising an N-terminal sortase recognition motif (e.g., a GGG, GG, or G motif, or a PEG-amine-terminated substrate) in a transpeptidation reaction. In some embodiments, the evolved sortases utilize a substrate different from those used by the parent sortase, e.g., substrates with a C-terminal LPXS, LPXSG (SEQ ID NO: 8), LAXT, LAXTG (SEQ ID NO: 9), MPXT, MPXTG (SEQ ID NO: 10), LAXS, LAXSG (SEQ ID NO: 11), NPXT, NPXTG (SEQ ID NO: 12), NAXT, NAXTG (SEQ ID NO: 13), NAXS, NAXSG (SEQ ID NO: 14), LPXP, LPXPG (SEQ ID NO: 15), or LPXTA (SEQ ID NO: 16) motif. In certain embodiments, the specificity of the evolved, new sortase has greater affinity for a particular C-terminal sortase recognition motif over another sequence, but the motif may also be recognized albeit less well by the wild-type sortase. Therefore, the specificity of the evolved sortase has been altered as compared to the wild-type sortase. In some embodiments, the specificity for a particular recognition motif is based on a comparison between the Km that the evolved sortase has for the motif, relative to that of the parent or wild type sortase. For example, in some embodiments, an evolved sortase has a K.sub.m for an altered (e.g., non-canonical or non-wild type motif) recognition motif that is at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 75-fold, at least 100-fold, at least 125-fold, at least 150-fold, at least 200-fold, at least 250-fold, at least 300-fold, at least 400-fold, at least 600-fold, at least 800-fold, or at least 1000-fold (or more) less than the K.sub.m that the parent sortase exhibits for the altered recognition motif.
(53) In some embodiments, a provided sortase comprises an amino acid sequence that is homologous to the amino acid sequence of a wild type sortase (e.g., to the amino acid sequence of S. aureus sortase A as provided as SEQ ID NO: 1), or a fragment thereof. In some embodiments, the amino acid sequence of the provided sortase comprises one or more mutations as compared to the wild type sequence of the respective sortase. For example, the evolved sortase sequence provided may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, or more mutations. In some embodiments, the sequence of the provided sortase is at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, or at least 99.5% identical to a wild type sortase sequence. In certain embodiments, the wild type sortase is S. aureus sortase A.
(54) In some embodiments, the evolved sortase comprises an S. aureus sortase A amino acid sequence, or a fragment thereof, with one or more of the following mutations: K84R, R99H or R99K, S102C, A104H, E105D, K138I or K138V or K138P, K145E, K152I, D160K, K162R or K162H, T164N, V168I, K177G or K177R, or I182F, and T196S. In some embodiments, the sortase includes at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten of the following mutations: K84R, R99H or R99K, S102C, A104H, E105D, K138I or K138V or K138P, K145E, K152I, D160K, K162R or K162H, T164N, V168I, K177G or K177R, or I182F, and T196S. In some embodiments, the sortase further includes one or more mutations selected from P94R, F122S, D124G, K134R, D160N, D165A, I182V, K190E, and K196T. In some embodiments, the sortase includes at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine of the following mutations: P94R, F122S, D124G, K134R, D160N, D165A, I182V, K190E, and K196T. In some embodiments, the sortase includes the mutations P94R, D160N, D165A, K190E, and K196T. In some embodiments, a sortase is provided that includes the mutations: K84R, P94R, F122S, D124G, K134R, K145E, D160N, K162R, D165A, V168I, K177G, I182F, K190E, and K196T. In some embodiments, a sortase is provided that includes the mutations: K84R, P94R, F122S, D124G, K134R, K145E, D160N, K162R, D165A, V168I, K177R, I182F, K190E, and K196T. In some embodiments, a sortase is provided that includes the mutations: P94R, D160N, K162R, D165A, V168I, I182F, K190E, and K196T. In some embodiments, a sortase is provided that includes the mutations: P94R, A104H, D160N, K162R, D165A, V168I, I182V, K190E, and K196T. In some embodiments, a sortase is provided that includes the mutations: P94R, R99H, A104H, K138I, D160N, K162R, D165A, I182V, K190E, and K196T. In some embodiments, a sortase is provided that includes the mutations: P94R, A104H, K138V, D160N, K162R, D165A, I182V, K190E, and K196T. In some embodiments, a sortase is provided that includes the mutations: P94R, R99K, A104H, K138V, D160K, K162R, D165A, I182V, K190E, and K196T. In some embodiments, a sortase is provided that includes the mutations: P94R, S102C, A104H, E105D, K138P, K152I, N160K, K162H, T164N, D165A, K173E, I182V, K190E, and T196S. In some embodiments, the sortases provided herein (e.g., as described in this paragraph) bind substrates comprising the sequence LAXT or LAXTG (SEQ ID NO: 9), wherein X represents any amino acid, for example a substrate comprising the sequence LAET (SEQ ID NO: 17) or LAETG (SEQ ID NO: 5).
(55) In some embodiments, the evolved sortase comprises an S. aureus sortase A amino acid sequence, or a fragment thereof, with one or more of the following mutations: N98D, S102C, A104V, A118S, F122A, K134G or K134P, and E189V, E189F, or E189P. In some embodiments, the sortase includes at least two, at least three, at least four, at least five, at least six, or at least seven of the following mutations: N98D, S102C, A104V, A118S, F122A, K134G or K134P, and E189V, E189F, or E189P. In some embodiments, the sortase further includes one or more mutations selected from P94R, N98S, A104T, A118T, F122S, D124G, K134R, D160N, D165A, I182V, K190E, and K196T. In some embodiments, the sortase includes at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least 11, or at least 12 of the following mutations: P94R, N98S, A104T, A118T, F122S, D124G, K134R, D160N, D165A, I182V, K190E, and K196T. In some embodiments, the sortase includes the mutations P94R, D160N, D165A, K190E, and K196T. In some embodiments, a sortase is provided that includes the mutations: P94R, N98S, A104T, A118T, F122S, K134R, D160N, D165A, K173E, K177E, I182V, K190E, and K196T. In some embodiments, a sortase is provided that includes the mutations: P94R, A104T, A118T, D160N, D165A, I182V, K190E, and K196T. In some embodiments, a sortase is provided that includes the mutations: P94R, A118T, F122S, D160N, D165A, I182V, K190E, and K196T. In some embodiments, a sortase is provided that includes the mutations: P94R, A104V, A118T, F122S, D160N, D165A, I182V, K190E, and K196T. In some embodiments, a sortase is provided that includes the mutations: P94R, N98D, A104V, A118T, F122A, K134R, D160N, D165A, I182V, K190E, and K196T. In some embodiments, a sortase is provided that includes the mutations: P94R, N98D, A104V, A118S, F122A, K134G, D160N, D165A, I182V, E189V, K190E, and K196T. In some embodiments, a sortase is provided that includes the mutations: P94R, N98D, A104V, A118S, F122A, K134P, D160N, D165A, I182V, E189V, K190E, and K196T. In some embodiments, a sortase is provided that includes the mutations P94R, N98D, S102C, A104V, A118T, F122A, K134R, F144L, D160N, D165A, I182V, E189F, K190E, and K196T. In some embodiments, the sortases provided herein (e.g., as described in this paragraph) bind substrates comprising the sequence, LPXS or LPXSG (SEQ ID NO: 8), wherein X represents any amino acid, for example, a substrate comprising the sequence LPES (SEQ ID NO: 18), LPESG (SEQ ID NO: 3). In some embodiments, the sortases provided herein bind substrates comprising the sequence LPXA or LPXC, such as LPEA or LPEC, respectively.
(56) In some embodiments, the evolved sortase provided herein comprises any of the following sets of amino acid mutations listed below: K84R, P94R, F122S, D124G, K134R, K145E, D160N, K162R, D165A, V168I, K177G, I182F, K190E, and K196T K84R, P94R, F122S, D124G, K134R, K145E, D160N, K162R, D165A, V168I, K177R, I182F, K190E, and K196T P94R, D160N, K162R, D165A, V168I, I182F, K190E, and K196T P94R, A104H, D160N, K162R, D165A, V168I, I182V, K190E, and K196T P94R, R99H, A104H, K138I, D160N, K162R, D165A, I182V, K190E, and K196T P94R, A104H, K138V, D160N, K162R, D165A, I182V, K190E, and K196T P94R, R99K, A104H, K138V, D160K, K162R, D165A, I182V, K190E, and K196T P94R, A104H, K138P, K152I, D160K, K162R, D165A, I182V, K190E, and K196T P94R, 5102C, A104H, E105D, K138P, K152I, N160K, K162H, T164N, D165A, K173E, I182V, K190E, and T196S.
In some embodiments, evolved sortases comprising the foregoing sets of amino acid mutations are those that bind substrates comprising LAXT.
(57) In some embodiments, the evolved sortase comprises any of the following sets of amino acid mutations listed below: P94R, N98S, A104T, A118T, F122S, K134R, D160N, D165A, K173E, K177E, I182V, K190E, and K196T P94R, N98S, A104T, A118T, F122S, D124G, K134R, D160N, D165A, K173E, K177E, I182V, K190E, and K196T P94R, A104T, A118T, D160N, D165A, I182V, K190E, and K196T P94R, A118T, F122S, D160N, D165A, I182V, K190E, and K196T P94R, A104V, A118T, F122S, D160N, D165A, I182V, K190E, and K196T P94R, A118T, F122S, D160N, D165A, I182V, K190E, and K196T P94R, N98D, A104V, A118S, F122A, D160N, D165A, I182V, K190E, and K196T P94R, N98D, A104V, A118T, F122A, K134R, D160N, D165A, I182V, K190E, and K196T P94R, N98D, A104V, A118S, F122A, K134G, D160N, D165A, I182V, E189V, K190E, and K196T P94R, N98D, A104V, A118S, F122A, K134P, D160N, D165A, I182V, E189V, K190E, and K196T P94R, N98D, S102C, A104V, A118T, F122A, K134R, F144L, D160N, D165A, I182V, E189F, K190E, and K196T
In some embodiments, evolved sortases comprising the foregoing sets of amino acid mutations are those that bind substrates comprising LPXS.
(58) In some embodiments, the evolved sortase comprises any of the sequences listed herein including those found in any figures or any tables found in the application. In any of the embodiments herein, the evolved sortases can comprise the mutation D160N or D160K. In any of the embodiments herein, the evolved sortases can comprise the mutation K196T or T196S.
(59) Some evolved sortases provided herein exhibit enhanced reaction kinetics, for example, they can achieve a greater maximum turnover per time unit (k.sub.cat) or a greater turnover per time under physiological conditions such as at a pH ranging from about 7.0 to about 7.6. For example, in some embodiments, an evolved sortase is provided herein that exhibits a k.sub.cat for an altered substrate recognition motif (e.g., LAXT or LPXS) that is at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 100-fold, or at least about 200-fold greater than the k.sub.cat of the corresponding wild type substrate recognition motif (e.g., LPXT).
(60) Some evolved sortases provided herein exhibit enhanced reaction specificities, e.g., in that they bind a substrate with higher affinity or with higher selectivity, or in that they bind a substrate that is not bound or not efficiently bound by the respective wild type sortase. For example, some sortases provided herein exhibit a K.sub.M for a substrate having an altered sortase recognition motif (e.g., LAXT or LPXS) that is at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, or at least about 50-fold less than the K.sub.M for the canonical or wild type sortase recognition motif (e.g., LPXT). Some evolved sortases provided herein, for example, exhibit a K.sub.M for a substrate comprising a C-terminal sortase recognition sequence of LAXT or LPXS that is at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, or at least about 15-fold less than the K.sub.M for a substrate comprising a C-terminal canonical or wild type sortase recognition sequence of LPXT.
(61) In some embodiments, evolved sortases are provided that bind one of their substrates (e.g., a substrate with a C-terminal sortase recognition motif) with a decreased K.sub.M while exhibiting no or only a slight decrease in the K.sub.M for the other substrate (e.g., a substrate with an N-terminal sortase recognition motif). For example, some evolved sortases provided herein exhibit a K.sub.M for a substrate comprising an N-terminal sortase recognition motif (e.g., GGG) that is not more than 2-fold, not more than 5-fold, not more than 10-fold, or not more than 20-fold greater than the K.sub.M of the corresponding wild type sortase (e.g., wild type S. aureus sortase A).
(62) In some embodiments, evolved sortases are provided herein that exhibit a ratio of k.sub.cat/K.sub.M for a substrate comprising an LAXT or LPXS sequence that is at least about 1.5-fold, at least about 2-fold, at least about 5-fold, at least about 10-fold, at least about 20-fold, at least about 50-fold, at least about 80-fold, at least about 100-fold, at least about 120-fold, at least about 150-fold, at least about 200-fold, at least about 300-fold, at least about 400 fold, at least about 500-fold, at least about 600-fold, at least about 700-fold, at least about 800-fold, at least about 900-fold, or at least about 1000-fold greater than the k.sub.cat/K.sub.M ratio for substrates comprising an LPXT sequence. In certain embodiments, a substrate comprising the sequence LAXT comprises the sequence LAET, and the substrate comprising the LPXT sequence comprises the sequence LPET. In certain embodiments, a substrate comprising the sequence LPXS comprises the sequence LPES, and the substrate comprising the sequence LPXT comprises the sequence LPET.
(63) In some embodiments, some of the evolved sortases provided exhibit increased stability compared to the wild-type or a parent sortase. For example, in some embodiments, the evolved sortases provided herein exhibit an increase in stability of T.sub.m of at least about 1.0 C., at least about 2.0 C., at least about 3.0 C., at least about 4.0 C., at least about 5.0 C., at least about 6.0 C., or at least about 10.0 C. compared to the stability of eSrtA. In some embodiments, the evolved sortases provided herein exhibit an increase in stability of T.sub.m of about 1.0 C. to about 10.0 C., 1.0 C. to about 3.0 C., 3.0 C. to about 5.0 C., 4.0 C. to about 5.0 C., 5.0 C. to about 8.0 C. compared to the stability of eSrtA. The stability of the evolved sortases can be determined using various methods in the art. For example, in certain embodiments, the stability can be determined using thermal melting curves as further described in the Examples below.
(64) Provided herein are sortases that can be used in orthogonal modification strategies. For example, a first sortase that has preferential activity for a first non-canonical substrate but not for a second non-canonical substrate can be used with a second sortase that has preferential activity for the second non-canonical substrate but not for the first non-canonical substrate. In some embodiments, evolved sortases are provided herein that exhibit a ratio of k.sub.cat/K.sub.M of about at least 100-fold, 200-fold, 300-fold, 400-fold, or 500-fold greater for a substrate comprising an LAXT sequence than for a substrate comprising an LPXT sequence and also exhibits negligible activity for another non-canonical substrate such as LPXS, wherein there is negligible activity when the sortase exhibits a ratio of k.sub.cat/K.sub.M for LPXS of about at least 1000-fold, at least 2000-fold, at least 3000-fold, at least 4000-fold, at least 5000-fold less than the ratio of k.sub.cat/K.sub.M for the LAXT. In certain embodiments, the LPXS-containing substrate comprises the sequence LPESG, the LPXT-containing substrate comprises the sequence LPETG, and the LAXT-containing substrate comprises the sequence LAETG. In certain embodiments, the evolved sortase comprises the mutations: P94R, S102C, A104H, E105D, K138P, K152I, N160K, K162H, T164N, D165A, K173E, I182V, K190E, and T196S and the evolved sortase exhibits a ratio of k.sub.cat/K.sub.M for LAETG of about 450-fold to 650-fold greater than the ratio of k.sub.cat/K.sub.M for LPETG. In certain embodiments, the evolved sortase also exhibits a ratio of k.sub.cat/K.sub.M for LPESG of about 5500-fold to 6500-fold less than the ratio for LPESG.
(65) In some embodiments, evolved sortases are provided herein that exhibit a ratio of k.sub.cat/K.sub.M of about at least 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, or 50-fold greater for a substrate comprising an LPXS sequence than for a substrate comprising an LPXT sequence and also exhibits negligible activity for another non-canonical substrate such as LAXT, wherein there is negligible activity when the sortase exhibits a ratio of k.sub.cat/K.sub.M for LAXT of about at least 100-fold, at least 200-fold, at least 300-fold, at least 400-fold, at least 500-fold, at least 600-fold, at least 700-fold, at least 800-fold less than the ratio of k.sub.cat/K.sub.M for the LPXS. In certain embodiments, the LPXS substrate comprises LPESG, the LPXT substrate comprises LPETG, and the LAXT substrate comprises LAETG. In certain embodiments, the evolved sortase comprises the mutations: P94R, N98D, S102C, A104V, A118T, F122A, K134R, F144L, D160N, D165A, I182V, E189F, K190E, and K196T, and the evolved sortase exhibits a ratio of k.sub.cat/K.sub.M for LPESG of about 20-fold to 30-fold greater than the ratio of k.sub.cat/K.sub.M for LPETG. In certain embodiments, the evolved sortase also exhibits a ratio of k.sub.cat/K.sub.M for LAETG of about 500-fold to 1000-fold less than the ratio for LPESG.
(66) In some embodiments, the evolved sortases provided herein exhibit a change in substrate specificity relative to eSrtA of about at least 50-fold, at least 100-fold, at least 200-fold, at least 500-fold, at least 1,000-fold, at least 5,000-fold, at least 10,000-fold, at least 20,000-fold, 30,000-fold, 40,000-fold, 50,000-fold, or 60,000-fold. An evolved sortase's change in substrate specificity relative to a parent sortase (e.g., eSrtA or the wild-type sortase) is determined by: dividing the evolved sortase's ratio of k.sub.cat/K.sub.M for a non-canonical substrate (e.g., LAXT) by the ratio of k.sub.cat/K.sub.M of a canonical substrate (e.g., LPXT) to obtain a first ratio; dividing the parent sortase's ratio of k.sub.cat/K.sub.M for a non-canonical substrate (e.g., LAXT) by the ratio of k.sub.cat/K.sub.M of a canonical substrate (e.g., LPXT) to get a second ratio; then dividing the first ratio by the second ratio. In some embodiments, the evolved sortases provided herein exhibit a change in substrate specificity relative to eSrtA of about 50-fold to about 150-fold. In some embodiments the change in substrate specificity is for a substrate comprising LPXS over LPXT. In some embodiments, the evolved sortases that exhibits about 50-fold to about 150-fold a change in substrate specificity relative to eSrtA is the 4S-9 variant. In some embodiments, the evolved sortases provided herein exhibit a change in substrate specificity relative to eSrtA of about 45,000-fold to about 55,000-fold. In some embodiments the change in substrate specificity is for a substrate comprising LAXT over LPXT. In some embodiments, the evolved sortases that exhibits about 45,000-fold to about 55,000-fold a change in substrate specificity relative to eSrtA is the 2A-9 variant.
(67) Methods of Use
(68) Some aspects of this invention provide methods for carrying out sortase-mediated transpeptidation reactions using the evolved sortases described herein. In some embodiments, such methods include contacting an evolved sortase provided herein with a suitable substrate, e.g., a substrate comprising a suitable C-terminal sortase recognition motif and a substrate comprising a suitable N-terminal sortase recognition motif under conditions suitable for sortase-mediated transpeptidation. In some embodiments, the evolved sortase is an evolved S. aureus sortase A carrying one or more of the mutations described herein. In some embodiments, the C-terminal sortase recognition motif is LAXT (e.g., LAETG; SEQ ID NO: 5), or LPXS (e.g., LPESG; SEQ ID NO: 3), and the N-terminal recognition motif is GGG.
(69) In some embodiments, at least one of the substrates is conjugated to a solid support. In some embodiments, at least one of the substrates is on the surface of a cell or other biological entity (e.g., virus). For example, in some embodiments, at least one of the sortase substrates is expressed as a fusion protein on the surface of a cell, e.g., a cell that expresses a surface marker protein that is C-terminally fused to an amino acid sequence comprising a C-terminal sortase recognition motif (e.g., LAXT, or LPXS), or that is N-terminally fused to an N-terminal sortase recognition motif (e.g., GGG).
(70) The transpeptidation reactions provided herein typically result in the creation of a protein fusion comprising the C-terminal sortase recognition motif and the N-terminal sortase recognition motif. In some embodiments, one of the substrates (e.g., the substrate comprising the C-terminal sortase recognition motif) comprises a non-protein structure, e.g., a detectable label, a small molecule, a nucleic acid, a polymer, or a polysaccharide. It will be apparent to those of skill in the art that the transpeptidation methods provided herein can be applied to conjugate any moieties that can be conjugated by any known sortase or sortase-mediated transpeptidation reaction, including, but not limited to, the reactions and moieties disclosed in Ploegh et al., International PCT Patent Application, PCT/US2010/000274, filed Feb. 1, 2010, published as WO/2010/087994 on Aug. 5, 2010; and Ploegh et al., International Patent Application PCT/US2011/033303, filed Apr. 20, 2011, published as WO/2011/133704 on Oct. 27, 2011; the entire contents of each of which are incorporated herein by reference, for exemplary sortases, proteins, recognition motifs, reagents, moieties, and methods for sortase-mediated transpeptidation. The invention is not limited in this respect.
(71) In some embodiments, methods for orthogonal protein modification, e.g., methods to modify a protein at either or both the N- and C-termini of a protein, are provided. For example, methods for N-terminal modification of a protein are provided. In some embodiments, the method involves contacting a protein comprising a N-terminal sortase recognition motif (e.g., GGG) with a sortase provided herein, and a modifying agent comprising a C-terminal sortase recognition motif (e.g., LPXT, LAXT, or LPXS), under conditions suitable for sortase-mediated transpeptidation. Methods for C-terminal protein modification typically involve contacting a protein comprising a C-terminal sortase recognition motif (e.g., LPXT, LAXT, or LPXS) with a sortase provided herein, and a modifying agent comprising a N-terminal sortase recognition motif (e.g., GGG), under conditions suitable for sortase-mediated transpeptidation. Methods for modifying a protein at both the N- and C-termini typically involve contacting the protein (sequentially or simultaneously) with two different sortases (e.g., sortases that bind different sortase recognition motifs), and two or more different modifying agents (or two or more modifying agents comprising the same agent but different sortase recognition motifs), under conditions suitable for sortase-mediated transpeptidation. For example, the method may involve contacting a protein comprising a N-terminal GGG motif and a C-terminal LAXT motif (under conditions suitable for sortase-mediated transpeptidation) with: (1) a provided sortase that binds and catalyzes transpeptidation reactions with substrates comprising the LAXT motif and a modifying agent comprising a GGG motif (thereby modifying the C-terminus of the protein); and (2) a provided sortase that binds and catalyzes transpeptidation reaction with substrates comprising the LPXS motif and a modifying agent comprising a LPXS motif (thereby modifying the N-terminus of the protein). Alternatively, the method may involve contacting a protein comprising a N-terminal GGG motif and a C-terminal LPXS motif (under conditions suitable for sortase-mediated transpeptidation) with: (1) a provided sortase that binds and catalyzes transpeptidation reactions with substrates comprising the LPXS motif and a modifying agent comprising a GGG motif (thereby modifying the C-terminus of the protein); and (2) a provided sortase that binds and catalyzes transpeptidation reaction with substrates comprising the LAXT motif and a modifying agent comprising a LAXT motif (thereby modifying the N-terminus of the protein). In some embodiments, either step (1) or step (2) in the above two examples may comprise using a protein or modifying agent with the wild type sortase A recognition motif (e.g., LPXT), and a sortase which binds and catalyzes transpeptidation reactions with substrates comprising the wild type recognition motif. In some embodiments, either step (1) or step (2) in the above two examples may comprise using a protein or modifying agent comprising a recognition motif from another sortase (e.g., sortase B, sortase C, or sortase D), and a sortase which binds and catalyzes transpeptidation reactions with such substrates. It should be appreciated, that other recognition motifs are amenable to these methods. The invention is not limited in this respect.
(72) Modifying agents include any of those described herein, and further include without limitation, polymers (e.g., artificial polymers such as polyethylene glycol (PEG) natural polymers such as nucleic acids, peptides, or proteins), carbohydrates (e.g., dextran), lipids, labels, radioisotopes, toxins, antibodies, solid surfaces, amino acids (including natural and non-natural amino acids), hormones (e.g., steroid hormones), enzyme cofactors, binding agents (e.g., biotin), chemical probes, and adjuvants.
(73) In another embodiment, methods for modifying a protein in (or on) a cell or tissue are provided. In some embodiments, the method involves contacting the protein in or on a cell or tissue (which comprises a sortase recognition motif) with a sortase provided herein, and a modifying agent comprising a sortase recognition motif under conditions suitable for sortase-mediated transpeptidation. In some embodiments, the protein comprises either or both a N-terminal sortase recognition motif (e.g., GGG) and/or a C-terminal recognition motif (e.g., LPXT, LAXT, or LPXS). In some embodiments, the protein comprises either a N-terminal sortase recognition motif (e.g., GGG) or a C-terminal recognition motif (e.g., LPXT, LAXT, or LPXS). In some embodiments, the modifying agent comprises either or both a N-terminal sortase recognition motif (e.g., GGG) and/or a C-terminal recognition motif (e.g., LPXT, LAXT, or LPXS), for example, as described herein. In some embodiments, the modifying agent comprises either a N-terminal sortase recognition motif (e.g., GGG) or a C-terminal recognition motif (e.g., LPXT, LAXT, or LPXS). In some embodiments, the protein either comprises a sortase recognition motif naturally (e.g., the wild type form of the protein comprises a sortase recognition motif as described herein) or is engineered to comprise a sortase recognition motif. Methods for engineering and expressing proteins in cells or tissue are well known in the art and include those as provided by, for example, Etcheverry, Expression of Engineered Proteins in Mammalian Cell Culture, in Protein Engineering: Principles and Practice, Cleland et al. (eds.), pages 163 (Wiley-Liss, Inc. 1996). In some embodiments, the modifying agents include any of those described herein, and further include without limitation, anti-clotting factors, immunotherapeutics, or anti-bacterial agents.
(74) Kits
(75) Some aspects of this invention provide kits including an evolved sortase (e.g., as described herein), and reagents useful for carrying out a transpeptidation reaction using the evolved sortase. For example, in some embodiments, the kit may comprise a nucleic acid encoding an amino acid sequence recognized by the sortase, e.g., a N-terminal or C-terminal sortase recognition motif (e.g. GGG, LAXT, LPXS), that can be used to generate protein fusions in which a protein of interest carries a desired recognition motif. In some embodiments, an enzyme substrate conjugated to a detectable label is included. In some embodiments, the kit includes a detectable label (e.g., biotin), a small molecule, a nucleic acid, a polypeptide, a polymer (e.g., PEG), or a polysaccharide (e.g., dextran). In some embodiments, the kit includes a radioisotope, a toxin, an antibody, or an adjuvant. In some embodiments, the kit includes buffers, for example, buffers comprising CaCl.sub.2. In some embodiments, the kit includes a buffer that buffers at physiological pH, e.g., at pH 7.4-pH7.6, for example, Tris HCl buffer pH 7.5. In some embodiments, the kit includes one or more vectors encoding a sortase provided herein, for example for use in recombinant protein expression or sortase-mediated modifications. In some embodiments, the kit includes cell overexpressing an evolved sortase provided herein.
(76) The function and advantage of these and other embodiments of the present invention will be more fully understood from the Examples below. The following Examples are intended to illustrate the benefits of the present invention and to describe particular embodiments, but are not intended to exemplify the full scope of the invention. Accordingly, it will be understood that the Examples are not meant to limit the scope of the invention.
EXAMPLES
(77) Materials and Methods
(78) Sortase Assay Methods
(79) In Vitro Sortase Kinetics Assays.
(80) See below for details on sortase expression and purification, and on the synthesis of Abz-LPETGK(Dnp)-CONH.sub.2 (SEQ ID NO: 19). Assays to determine k.sub.cat and K.sub.m LPETG, K.sub.m LAETG and K.sub.m LPESG were performed in 300 mM Tris pH 7.5, 150 mM NaCl, 5 mM CaCl.sub.2, 5% v/v DMSO, and 9 mM Gly-Gly-Gly-COOH (GGG). The concentration of the LPETG (SEQ ID NO: 4), LAETG (SEQ ID NO: 5), and LPESG (SEQ ID NO: 3) peptide substrates ranged from 12.5 M to 10 mM, and enzyme concentrations ranged from 25 nM to 1000 nM. Assays for determination of K.sub.m GGG were performed under the same conditions, except the LPETG (SEQ ID NO: 4), LAETG (SEQ ID NO: 5), and LPESG (SEQ ID NO: 3) peptide concentrations were fixed at 1 mM, the enzyme concentration was fixed at 41.5 nM, and the concentration of GGG was varied from 33 M to 30 mM, depending on the enzyme. Reactions were initiated with the addition of enzyme and incubated at 22.5 C. for 3 to 20 minutes before quenching with 0.5 volumes of 1 M HCl. Five to ten nmol of peptide from the quenched reactions were injected onto an analytical reverse-phase Eclipse XDB-C18 HPLC column (4.6150 mm, 5 m, Agilent Technologies) and chromatographed using a gradient of 10 to 65% acetonitrile with 0.1% TFA in 0.1% aqueous TFA over 13 minutes. Retention times under these conditions for the Abz-LPETGK(Dnp)-CONH.sub.2 (SEQ ID NO: 19) substrate, the released GKDnp peptide, and the Abz-LPETGGG-COOH (SEQ ID NO: 20) product were 12.8, 10.4, and 9.1 min, respectively. To calculate the percent conversion, the ratio of the integrated areas of the Abz-LPETGGG-COOH (SEQ ID NO: 20) and Abz-LPETGK(Dnp)-CONH.sub.2 (SEQ ID NO: 19) peptide Abs220 peaks was compared to a standard curve generated by mixing the product and starting peptide in known ratios. To determine k.sub.cal and K.sub.m, reaction rates were fit to the Michaelis-Menten equation using OriginPro 7.0 software. All kinetics values reported represent the average of at least three measurements. Percent conversion was also calculated by using the ratio of the integrated areas of the GK(Dnp)-CONH2 and Abz-LPETGK(Dnp)-CONH2 peptide Abs355 peaks were compared directly (Example 5). To determine k.sub.cat and K.sub.m, LPETG, reaction rates were fit to the Michaelis-Menten equation using Microsoft Excel using the Solver add-in. To determine K.sub.m,GGG and K.sub.H, reaction rates were fit to the modified Michaelis-Menten, for which K.sub.H is as defined when K.sub.H<<K.sub.m,GGG.
(81) Substrate Synthesis Methods
(82) Biotin-LC-LELPETGG-CONH.sub.2 (SEQ ID NO: 21), Fmoc-GGGK-CONH.sub.2 (SEQ ID NO: 22), and NH.sub.2YLELPETGG-CONH.sub.2 (SEQ ID NO: 23) were purchased from Genscript and used without further purification. NH.sub.2-GGGYK(biotin)-CONH.sub.2 (SEQ ID NO: 24) was purchased from Genscript and purified using reverse-phase HPLC on a C18 column. Biotin-LCYGLPETGS-CONH.sub.2 (SEQ ID NO: 25) was purchased from New England Peptide and used without further purification.
(83) Synthesis of GGGK-CoA.
(84) Fmoc-GGGK-CONH.sub.2 (SEQ ID NO: 22) was dissolved in DMSO to a final concentration of 100 mM, and 1.5 equivalents of sulfo-SMCC (Thermo-Fisher) and 2 equivalents of DIPEA (Sigma) in DMSO were added. The reaction was incubated for 1 hr at room temperature, then added to 1.5 equivalents of coenzyme A trilithium hydrate (Sigma) in DMSO to a final peptide concentration of 25 mM and mixed at room temperature overnight. If appropriate, the Fmoc protecting group was removed with 20% vol/vol piperidine and incubation for 20 minutes. The reaction was quenched by the addition of 1 equivalent of TFA, and the product was purified on a preparative Kromasil 100-5-C18 column (21.2250 mm, Peeke Scientific) by reverse phase HPLC (flow rate: 9.5 mL/min; gradient: 10% to 70% acetonitrile with 0.1% TFA in 0.1% aqueous TFA gradient over 30 minutes; retention time: 17.1 minutes). ESI-MS (found): [M-H] m/z=1300.1. Calculated for C45H72N14O23P3S: m/z=1301.4. The concentration of GGGK-CoA (SEQ ID NO: 22) peptide was determined from the measured A259 using the known molar extinction coefficient of coenzyme A, 15,000 M.sup.1 cm.sup.1 (Killenberg P G & Dukes D F (1976) Coenzyme A derivatives of bile acids-chemical synthesis, purification, and utilization in enzymic preparation of taurine conjugates. J Lipid Res 17(5):451-455).
(85) Synthesis of CoA-LPETGG
(86) NH.sub.2YLELPETGG-CONH.sub.2 (SEQ ID NO: 26; SEQ ID NO: 23)(0.0084 mmol) was incubated with sulfo-SMCC (0.021 mmol, 2.5 eq.) in 142 L of DMSO and 3 L DIPEA (0.017 mmol, 2.0 equivalents) for 2 hours at room temperature. The maleimide adduct was purified using reverse-phase HPLC on a preparative C18 column (flow rate: 9.5 mL/min; gradient: 10% to 60% acetonitrile with 0.1% TFA in 0.1% aqueous TFA over 30 minutes; retention time: 22.0 minutes). After lyophilization of the collected peak, the white solid was dissolved in 0.1 M phosphate buffer pH 7.0 with 45% acetonitrile. Coenzyme A trilithium hydrate (11.2 mg) was added, and the reaction was incubated at one hour at room temperature. The desired product was obtained after purification on a C18 column (flow rate: 9.5 mL/min flow rate; 0% to 50% acetonitrile in 0.1 M triethylammonium acetate over 30 minutes; retention time: 21.9 minutes). ESI-MS (found): [M-H] m/z=1961.8. Calculated for C.sub.77H.sub.116N.sub.18O.sub.34P.sub.3S: m/z=1961.7. The concentration of CoA-LPETGG (SEQ ID NO: 26) peptide was determined as described above for GGGK-CoA (SEQ ID NO: 22).
(87) Abz-L(A/P)E(T/S)GK(Dnp)-CONH.sub.2 Substrate for HPLC Assays (SEQ ID NO: 19).
(88) Each compound was synthesized at 200 mol scale using an Applied Biosystems 433A peptide synthesizer. 200 mol-equivalents of NovaPEG Rink Amide resin (EMD biosciences) were loaded onto the machine and coupled using 5 equivalents of each Fmoc-protected amino acid building block with standard acid labile side-chain protecting groups (Thr(OtBu), Glu(OtBu)) and using Fmoc Lysine(Dnp) (Chem-Impex). Terminal coupling with Boc 2-Aminobenzoic Acid (Chem-Impex) yielded the fully protected peptide, which was cleaved by three 1-hour treatments with 20 mL of 95% TFA+2.5% water+2.5% triisopropylsilane (Sigma). The cleavage mixtures were pooled and concentrated by rotary evaporation, and the peptide was precipitated by the addition of 9 volumes of ice-cold diethyl ether. The samples were purified by reverse phase HPLC as described above for GGGK-CoA (SEQ ID NO: 22)(retention time: 28 minutes), pooled and concentrated by lyophilization. The concentration of the peptide was determined by the known molar extinction coefficient of the Dnp group, f355 nm=17,400 M-1 cm-1 (Carsten M E & Eisen H N (1953) The Interaction of Dinitrobenzene Derivatives with Bovine Serum Albumin. Journal of the American Chemical Society 75(18):4451-4456).
(89) Alexa Fluor 750-LAETG Synthesis (SEQ ID NO: 5).
(90) 25 mg Alexa Fluor 750 NHS Ester was dissolved in 45 L of 0.4 M H2N-LAETGG peptide in DMSO and incubated at room temperature for 6 hours. 2.5 L DIPEA was added and incubated at room temperature overnight. Reactions were quenched by the addition of 450 L 1 M Tris, pH 7.5, and were incubated on ice for 2 hours. This reaction was purified on a preparative Kromasil 100-5-C18 column (21.2250 mm, Peeke Scientific) by reverse phase HPLC (flow rate: 9.5 mL/min; gradient: 10% to 70% acetonitrile with 0.1% TFA in 0.1% aqueous TFA gradient over 30 minutes; retention time 8 minutes) before pooling and lyophilizing the collected fractions. The concentration of the peptide was determined by the known molar extinction coefficient of Alexa Fluor 750 (Berlier, J. E. et al. Quantitative comparison of long-wavelength Alexa Fluor dyes to Cy dyes:fluorescence of the dyes and their bioconjugates. Journal of Histochemistry & Cytochemistry 51, 1699-1712 (2003)), 749 nm=290,000 M-1 cm-1.
(91) Alexa Fluor 488-LAETG (SEQ ID NO: 5) and Alexa Fluor 647-LPESG (SEQ ID NO: 3) Synthesis.
(92) To prepare Alexa Fluor 488-LAETG (SEQ ID NO: 5), 1 mg Alexa Fluor 488 NHS Ester and 3.33 mg Ac-KLAETGG (SEQ ID NO: 35) peptide were dissolved in 200 L of in DMF and incubated at room temperature for 1 hour. 5 L DIPEA was added and incubated at room temperature overnight. Reactions were quenched by the addition of 2 mL of 1 M Tris, pH 7.5, and incubated on ice for 2 hours. The products were purified on a preparative C18 column (1015-mm, varian) by reverse phase HPLC (flow rate: 5 mL/min; gradient: 5% to 50% acetonitrile with 0.1% TFA in 0.1% aqueous TFA gradient over 30 minutes) before pooling and lyophilizing the collected fractions. Alexa Fluor 647 NHS Ester and Ac-KLPESGG (SEQ ID NO: 37) peptides were similarly combined to generate Alexa Fluor 647-LPESG (SEQ ID NO: 3).
(93) GGG-PEG Synthesis.
(94) 100 mg 10 kDa PEG-NH2, 10 kDa bis-PEG-NH2, 10 kDa 4-arm-PEG-NH2, 5 kDa or 10 kDa Biotin-PEG-NH2 was dissolved in 500 L dry dichloromethane. 250 L of a slurry of 164 mg Fmoc-Gly-Gly-Gly-COOH (BAChem), 132 mg of HATU, and 56 mg of HOAt dissolved in 1 mL dry DMF were added. The resulting mixture was sonicated for 20 minutes before the addition of 35 L DIPEA, then sonicated an additional 20 minutes before incubation at room temperature for 16 hours. The mixture was quenched on ice by the addition of 100 L trifluoroacetic acid (TFA) then precipitated by addition to 10 mL of cold diethyl ether and recrystallized twice from warm, 100% ethanol. This material was filtered, dried under reduced pressure, then taken up in 1 mL 20% piperidine in dichloromethane and incubated at room temperature for 30 minutes to remove the Fmoc group. The reaction was quenched by the addition of 1 mL TFA on ice, precipitated with ethanol, and then recrystallized twice from warm 100% ethanol.
(95) pET29 Sortase Expression Plasmids
(96) Sortase genes were subcloned into pET29 at NdeI and XhoI using the primers 5F and 5R. Plasmids encoding sortase single mutants were constructed using the Quikchange method. All expressed sortases lack the N-terminal 59 amino acids.
(97) Protein Expression and Purification
(98) Bacterial Expression of Sortases. E. coli BL21 (DE3) transformed with pET29 sortase expression plasmids were cultured at 37 C. in LB with 50 g/mL kanamycin until OD600=0.5-0.8. IPTG was added to a final concentration of 0.4 mM and protein expression was induced for three hours at 30 C. The cells were harvested by centrifugation and resuspended in lysis buffer (50 mM Tris pH 8.0, 300 mM NaCl supplemented with 1 mM MgCl.sub.2, 2 units/mL DNAseI (NEB), 260 nM aprotinin, 1.2 M leupeptin, and 1 mM PMSF). Cells were lysed by sonication and the clarified supernatant was purified on Ni-NTA agarose following the manufacturer's instructions. Fractions that were >95% purity, as judged by SDS-PAGE, were consolidated and dialyzed against Tris-buffered saline (25 mM Tris pH 7.5, 150 mM NaCl). Enzyme concentration was calculated from the measured A280 using the published extinction coefficient of 17,420 M-1 cm-1 (Kruger R G, et al. (2004) Analysis of the substrate specificity of the Staphylococcus aureus sortase transpeptidase SrtA. Biochemistry 43(6):1541-1551).
(99) N- and C-terminal Protein modification using Evolved Sortases
(100) 10 M purified FGF2 or FGF21 and/or 5 M of a candidate Sortase and either 100 M GGG-Biotin or 100 uM Btn-LPESG (SEQ ID NO: 3) was/were taken up in 100 mM Tris buffer, pH 7.5, 500 mM NaCl, 5 mM CaCl2 and incubated for an hour at room temperature. Reactions were quenched by the addition of 4SDS loading buffer (cf 2% SDS, 10% Glycerol, 5% B-Mercaptoethanol, 2.5 mM EDTA), heated in PCR strips to 95C for 10 minutes, then put on ice and immediately run on NuPage 4-12% Bis-Tris gels with MES running buffer, blotted to PDME and visualized by anti-His6/anti-Mouse 680 (SEQ ID NO: 6) and Streptavidin-800. The experimental design and results are illustrated in
(101) Reactions were performed as described in the previous section, with varying concentrations of sortase relative to FGF (1 eq=10 uM, 0.5 eq=5 uM, 0.2 eq=2 uM, 0.1 eq=1 uM) and with 200 uM GGG-Biotin. Results are illustrated in
(102) Tissue Modification using Evolved Sortases
(103) Tissue section microarrays were purchased from the BioChain institute (Cat. Z7020001, Lot B508112) and deparaffinized by treatment with 2 changes Xylene for 9 minutes each, 1 change 1:1 Xylene:Ethanol for 3 minutes, 1 change 100% ethanol for 3 minutes, 1 change 90% ethanol for 3 minutes, 1 change 70% ethanol for 3 minutes, 1 change 70% ethanol+0.25% ammonia for 1 hour, 1 change 50% ethanol for 3 minutes, 1 change DI water for 5 minutes. Antigen retrieval was achieved by soaking in 10 mM Citrate pH 6 followed by microwave heating for 2 minutes, then let to rest for 8 minutes. Tissues were washed with 10 mg/mL freshly prepared Sodium borohydride in PBS for 30 minutes on ice, then washed twice with PBS for 3 minutes each.
(104) Slides were blocked in 5% BSA/PBS for 1 hour at room temperature, then 0.001% (w/v) avidin in 5% BSA/PBS for 15 minutes, washed with 5% BSA/PBS, 0.001% (w/v) biotin in 5% BSA/PBS for 15 minutes, washed with 5% BSA/PBS and washed twice in TBS-BC (100 mM TBS, 500 mM NaCl, 0.5% BSA, 5 mM CaCl2) for three minutes. Slides were then treated with 20 uM Enzyme and 200 uM GGG-Biotin for 1 hour at room temperature, washed with 10 mM GGG in 5% BSA/PBS for 3 minutes, then washed twice with 5% BSA/PBS for 3 minutes. Slides were then treated with 2 mg/mL Sudan Black B in 70% EtOH for 30 minutes, washed three times with 5% BSA/PBS, incubated in 5% BSA/PBS+1:250 Streptavidin-568 for 30 minutes, washed 3 times with PBS, incubated with DAPI/PBS for 30 minutes and finally washed 3 times with PBS. Slides were dried, mounted and then visualized by confocal microscopy.
(105) 750-LAETG-FGF-LPESG-PEG Dual Labeling Protocol.
(106) SUMO-TEV-FGF-LPESG-His6 (SEQ ID NO: 27) conjugates were treated with 0.2 eq eSrtA(4S-9), 1 mM GGG-PEG and 10 mM CaCl2 in TBS and incubated at room temperature at 500 L final volumes. These samples were then quenched by the addition of 100 M H2N-LPESGG (SEQ ID NO: 38) peptide and 100 L of pre-equilibrated Ni-NTA resin slurry in TBS, then incubated on ice for 15 minutes. This mixture was then passed through a 0.2 m spin filter, diluted 1:10 into PBS+10 M H2N-LPESGG (SEQ ID NO: 38) peptide, and concentrated against a 10 kDa MWCO spin concentrator to a final volume of 400 L. This process was repeated five times to afford the crude SUMO-TEV-FGF-LPESGGG-PEG (SEQ ID NO: 39) conjugates. These conjugates were then co-treated with 0.5 eq TEV protease, 0.2 eq eSrtA(2A-9), 1 mM 750-LAETG and 10 mM CaCl2 in TBS for 1 hour, then subjected to an identical purification process. This crude sample was separated into >30 kDa fractions and <30 kDa fractions by a 30 kDa MWCO spin concentrator to provide the conjugates in good purity. Protein concentrations were determined by BCA assay.
(107) Plasma Labeling of Fetuin A.
(108) Normal human plasma was purchased from VWR (part number 89347-902) and stored at 20 C. For all reactions, an aliquot was thawed at 37 C. for 15 minutes, then vortexed to resuspend any coagulated material. To this sample was added 2 L of 0.1 M GGGK(biotin) (SEQ ID NO: 22) for analytical reactions. Higher concentrations of GGGK(biotin) (SEQ ID NO: 22) were avoided for analytical purposes, as they caused increased background in downstream Western blotting. 10 L of 1 M CaCl2 was added in cases of calcium supplementation. Reactions were initiated by the addition of 10 L of 100 M eSrtA, eSrtA(2A-9), or eSrtA(4S-9) and incubated at room temperature for 2 hours. Reactions were quenched by 10:1 dilution into SDS-PAGE loading buffer, followed by a 10 minute incubation at 95 C. These samples were then run on 4-12% Bis-Tris PAGE gels, blotted to PVDF membrane using the iBlot2 dry blotting system, blocked with Pierce Superblock buffer for 1 hour followed by incubation with 1:500 dilutions of Abcam -Fetuin A antibody (MM0273-6M23) in Superblock buffer with 0.1% Tween-20 for 1 hour. The blot was then washed 3 times with PBS+0.1% Tween-20, then incubated again in 1:15,000 LiCor goat antimouse 680+1:15,000 Licor Streptavidin-800 in Superblock buffer with 0.1% Tween and 0.01% SDS added for 45 minutes. The blot was then washed 3 times with PBS+0.1% Tween and visualized on an Odyssey IR imager.
(109) Materials Functionalization.
(110) GGG-functionalized substrate was generated by incubating 100 L of 2 M biotin-PEG (5K)-GGG in streptavidin coated 96 well microplates (Pierce) for 2 hours at room temperature, and washed three times with TBST buffer (20 mM Tris, 100 mM NaCl, pH 7.5 with 0.05% Tween 20). GGG-well plates were reacted at room temperature with 100 L of solutions containing sortase enzymes (50 nM each) and fluorophore-linked peptides (2.5 M). For orthogonal specificity tests, Alexa Fluor 488-LAETGG (SEQ ID NO: 35), Alexa Fluor 647-LPESGG (SEQ ID NO: 38), or both were reacted with eSrt(2A-9), eSrt(4S-9), or both in the presence of 100 mM of CaCl2 in 25 mM Tris, 500 mM NaCl, pH 7.5. After 2 hours, the well plates were washed with TBST three times, and TBS buffer was added to each well. Total fluorescent intensities were measured at 488 nm and 647 nm using a Biotek Synergy NEO HTS Multi Mode microplate reader. Experiments from three independents replicates were averaged, and are shown in
(111) Yeast Library Construction.
(112) Fresh plates of ICY200 S. cerevisiae cells were streaked from long-term glycerol stocks and grown for 72 hours at 30 C. prior to use. A single colony was picked and grown in 10 mL YPD+100 U/mL penicillin, 100 g/mL streptomycin, 100 g/mL kanamycin overnight with shaking at 30 C. This suspension culture was freshly diluted into 125 mL YPD and electrocompetent cells were prepared as described by Chao, G. et al. (solating and engineering human antibodies using yeast surface display. Nature protocols 1, 755-768 (2006). All library transformations were performed by gap repair homologous recombination into pCTCon2CTev vectors linearized by NheI and BamHI digestion.
(113) Yeast Library.
(114) Induction Libraries were grown in SCD-Trp-Ura dropout media+100 U/mL penicillin, 100 g/mL streptomycin, 100 g/mL kanamycin at 30 C. Library expression was induced by transfer to SGR-Trp-Ura media at 20 C. overnight.
(115) Yeast Contamination Removal.
(116) Periodically, S. cerevisiae cultures become contaminated with an unknown fungal growth, believed to originate from airborne spores. To remove the contaminant, we used a strategy of physical separation for rounds 4-9 cultures. Whenever contamination was observed, each outgrowth of library material was centrifuged at 400g for 30 minutes in a spinning bucket rotor against a step gradient of 30%, 27.5% and 25% Ficoll-400PM in PBS. Under these conditions, S. cerevisiae cells pelleted efficiently while contaminating organisms remained in the lower-density layers of the Ficoll gradient.
(117) Library Subcloning and Gene Isolation.
(118) Following selections, yeast were grown to saturation (OD 1.5) in SCD-Trp-Ura dropout media+100 U/mL penicillin, 100 g/mL streptomycin, 100 g/mL kanamycin at 30 C., then lysed using a Zymo Research Zymoprep II kit according to manufacturer's instructions. Harvested plasmid was then either transformed directly into NEB One-Shot Chemically Competent Top10 cells according to manufacturer's instructions, or amplified by PCR using external primers pCTCon2CTEV.HR2.Fwd and pCTCon2CTEV.HR2.Rev, purified by gel electrophoresis, and then either mutated directly for another round of selection or digested with BamHI and XhoI and ligated into pre-digested pET29B vector, then transformed into NEB One-Shot Chemically Competent NEBTurbo cells.
(119) TABLE-US-00002 pCTCon2CTEV.HR2.Fwd(SEQIDNO:40): CCCATACGACGTTCCAGACTATGCAGGATCTGAGAACTTGTACTTT CAAGGTGCT pCTCon2CTEV.HR2.Rev(SEQIDNO:41): CTGTTGTTATCAGATCTCGAGCTATTACAAGTCCTCTTCAGAAATA AGCTTTTGTTCGGA
Chemical Mutagenesis Library Construction (Rounds 1-3).
(120) Libraries were synthesized as described in Chen et al., PNAS, 108(28):11399-404 (2011). In short, genes isolated from yeast libraries were subcloned into Invitrogen chemically competent TOP10 cells, grown in 25 mL LB+50 g/mL carbenicillin, harvested using a Zymo Research Zymoprep II kit according to manufacturer's instructions and then mutagenized by PCR reactions containing 5 M 8-oxo-2deoxyguanosine (8-oxodGTP), 5 M 6-(2-deoxy-b-D-ribofuranosyl)-3,4-dihydro-8H-pyrimido-[4,5-C][1,2]oxazin-7-one (dPTP), 200 M each dNTP, and 0.4 M each of primers pCTCon2CTEV.HR2.Fwd and pCTCon2CTEV.HR2.Rev. PCR reactions were thermocycled ten times and the mutagenized genes were further amplified in PCR reactions without mutagenic dNTP analogs using the same primers. Gel-purified genes were combined with NheI/BamHI-digested pCTCon2CTev vectors in a 5:1 insert:backbone mass ratio and electroporated into ICY200 as previously described by Chao et al. Isolating and engineering human antibodies using yeast surface display. Nature protocols 1, 755-768 (2006) to yield the listed library sizes.
(121) Site Saturation Mutagenesis Library Construction (Rounds 4-6).
(122) Genes were amplified from harvested yeast libraries by PCR using the primers pET29.SrtA.Fwd and pET29.SrtA.Rev, purified by gel electrophoresis and digested using restriction enzymes XhoI and BamHI. These inserts were then ligated into pre-digested pET29B vectors, and then transformed into Life Technologies One Shot Mach1 cells, grown overnight in 25 mL LB+25 g/mL kanamycin, and then harvested to afford the subcloned library. 100 ng of this material was then subjected to PCR amplification with either of two primer pairs encoding an NNK randomization codon (round 4, 2A Library: 104Fwd/104Rev, 168+182Fwd/168Rev; round 4, 4S Library: 104+118Fwd/104Rev, 182Fwd/182Rev; round 5, 2A Library: 162+168Fwd/162Rev, 182Fwd/182Rev; round 5, 4S Library: 118+122Fwd/118Rev, 182Fwd/182Rev; round 6, 2A Library: 99+138Fwd/99Rev, one of 160Fwd/160Rev, 165Fwd/165Rev, 189Fwd/189Rev, 190Fwd/190Rev or 196Fwd/196Rev; round 6, 4S Library: 132Fwd/132Rev, one of 160Fwd/160Rev, 165Fwd/165Rev, 189Fwd/189Rev, 190Fwd/190Rev or 196Fwd/196Rev). PCR products were purified by gel electrophoresis, treated with the NEBNext End Repair Module according to manufacturers' instructions, blunt-end ligated using NEB Quick Ligase according to manufacturers' instructions, and then cloned into Life Technologies One Shot Mach1 cells. The resulting cells were grown overnight in liquid culture with 25 g/mL kanamycin and then harvested to afford the semirandom library. This procedure was repeated for the other randomization primer set in a given pair. Both products were pooled and subsequently mutagenized by using the Stratagene Mutazyme II DNA mutagenesis kit for 25 cycles of PCR amplification using primers pCTCon2CTEV.HR2.Fwd and pCTCon2CTEV.HR2.Rev. PCR reactions were purified by spin column and combined with NheI/BamHI-digested pCTCon2CTev vectors in a 5:1 insert:backbone mass ratio, and electroporated into ICY200 as previously described to yield the listed library sizes. Primer Sequences (SEQ ID NO: 42-63 from left to right and then top to bottom, respectively):
(123) TABLE-US-00003 104Fwd NNKgaagaaaatgaatcactagatgatcaaaatatttc 104Rev aaagcttacacctctatttaattgttcagatgttgc 168+ 182Fwd NNKctagatgaacaaaaaggtaaagataaacaattaacatt aNNKacttgtgatgattacaatgaagagacaggcgtttg 168Rev ttctacagctgttggcttaacatttcttatacttg 104+ 118Fwd NNKgaagaaaatgaatcactagatgatcaaaatatttcaat tNNKggacacactttcattgaccgtccgaactatc 104Rev aaagcttacacctctatttaattgttcagatgttgc 182Fwd NNKacttgtgatgattacaatgaagagacaggcgtttg 182Rev taatgttaattgtttatctttacctttttgttc 99+ 138Fwd: gcaggacacactttcattgaccgtccgaactatcaatttac aaatcttaaagcagccaaaNNKggtagtatggtgtacttta aagaggtaatg 99Rev: aattgaaatattttgatcatctagtgattcattttcttcat gaaagcttacaccMNNatttaattgttcagatgagctggtc ctggatatac 132Fwd NNKcttaaagcagccaaaaaaggtagtatggtgtac 132Rev tgtaaattgatagttcggacggtcaatgaaagtg 160Fwd NNKaagccaacagctgtagaagttctagatgaacaaaaag 160Rev atttcttatacttgtcattttatacttacgtg 165Fwd NNKgtagaagttctagatgaacaaaaaggtaaag 165Rev tgttggcttaacatttcttatacttgtcattttatac 189Fwd NNKgagacaggcgtttgggaaactcgtaaaatctttg 189Rev attgtaatcatcacaagtaattaatgttaattg 190Fwd NNKacaggcgtttgggaaactcgtaaaatctttgtag 190Rev ttcattgtaatcatcacaagtaattaatgttaattg 196Fwd NNKcgtaaaatctttgtagctacagaagtcaaactc 196Rev ttcccaaacgcctgtctcttcattgtaatcatc
Mutagenic PCR Library Construction (Rounds 7-9).
(124) Genes were isolated from harvested yeast libraries by PCR using the primers pCTCon2CTEV.HR2.Fwd and pCTCon2CTEV.HR2.Rev, purified by gel electrophoresis, and subsequently mutagenized by using the Stratagene Mutazyme II DNA mutagenesis kit for 25 cycles of PCR amplification using primers pCTCon2CTEV.HR2.Fwd and pCTCon2CTEV.HR2.Rev. Reactions were purified by spin column and combined with NheI/BamHI-digested pCTCon2CTev vectors in a 5:1 insert:backbone mass ratio and electroporated into ICY200 as described to yield the listed library sizes.
(125) TEV Protease Expression and Purification
(126) E. coli BL21 (DE3) harboring the pRK793 plasmid for TEV S219V expression and the pRIL plasmid (Addgene) was cultured in LB with 50 g/mL carbenicillin and 30 g/mL chloramphenicol until OD600 0.7. IPTG was added to a final concentration of 1 mM, and the cells were induced for three hours at 30 C. The cells were pelleted by centrifugation and lysed by sonication as described above. The clarified lysate was purified on Ni-NTA agarose, and fractions that were >95% TEV S219V were consolidated and dialyzed against TBS. Enzyme concentrations were calculated from A280 measurements using the reported extinction coefficient (Kapust, R. B. et al. Tobacco etch virus protease: mechanism of autolysis and rational design of stable mutants with wild-type catalytic proficiency. Protein engineering 14, 993-1000 (2001)) of 32,290 M-1 cm.-1
(127) Sortase Expression and Purification.
(128) E. coli BL21 (DE3) transformed with pET29 sortase expression plasmids were cultured at 37 C. in LB with 50 g/mL kanamycin until OD600=0.5-0.8. IPTG was added to a final concentration of 0.4 mM and protein expression was induced for three hours at 30 C. The cells were harvested by centrifugation and resuspended in lysis buffer (50 mM Tris pH 8.0, 300 mM NaCl supplemented with 1 mM MgCl2, 2 units/mL DNAseI (NEB), 260 nM aprotinin, 1.2 M leupeptin, and 1 mM PMSF). Cells were lysed by sonication and the clarified supernatant was purified on Ni-NTA agarose following the manufacturer's instructions. Fractions that were >95% purity, as judged by SDS-PAGE, were consolidated and dialyzed against Tris-buffered saline (25 mM Tris pH 7.5, 150 mM NaCl). Enzyme concentrations were calculated from A280 measurements using the published extinction coefficient (Kruger, R. G. et al. Analysis of the Substrate Specificity of the Staphylococcus aureus Sortase Transpeptidase SrtA. Biochemistry 43, 1541-1551, doi:10.1021/bi035920j (2004)) of 17,420 M-1 cm-1.
(129) FGF Expression and Purification
(130) Codon-optimized FGF1 and FGF2 constructs were synthesized as gBlocks from Integrated DNA Technologies. These genes were cloned via restriction digestion and ligation into pET29 expression plasmids with similarly optimized SUMO-TEV Cleavage site and LPESG- (SEQ ID NO: 3) linkers at their N- and C-termini, respectively. E. coli BL21 (DE3) transformed with these plasmids were cultured at 37 C. in LB with 50 g/mL kanamycin until OD600=0.5-0.8. IPTG was added to a final concentration of 0.4 mM and protein expression was induced for three hours at 30 C. The cells were harvested by centrifugation and resuspended in lysis buffer (50 mM Tris pH 8.0, 300 mM NaCl supplemented with 1 mM MgCl2, 2 units/mL DNAseI (NEB), 260 nM aprotinin, 1.2 M leupeptin, and 1 mM PMSF). Cells were lysed by sonication and the clarified supernatant was purified on Ni-NTA agarose following the manufacturer's instructions. Fractions that were >95% purity, as judged by SDS-PAGE, were consolidated and dialyzed against Tris-buffered saline (25 mM Tris pH 7.5, 150 mM NaCl). Protein concentration was calculated by BCA assay.
(131) Site-Directed Mutagenesis for eSrtA(2A-9) and eSrtA(4S-9) Point Mutant Cloning
(132) Sortase pET29 expression plasmids for eSrtA(2A-9) or eSrtA(4S-9) underwent around the world PCR with designed primers containing a single amino acid mutation. PCR reactions were treated with 1 L DpnI for 1 h at 37 C., then purified by gel electrophoresis, blunt-end ligated using NEB Quick Ligase according to manufacturers' instructions, and then cloned into Life Technologies One Shot Mach1 cells. Resulting colonies were grown in LB+50 g/mL kanamycin and plasmid DNA was harvested via miniprep kit.
(133) eSrtA(2A-9) Primer Sequences (SEQ ID NOs: 64-67 from left to right and then top to bottom, respectively):
(134) TABLE-US-00004 H104A-Fwd 5Phos/GCGgacgaaaacgaaagcctggatg H104A-Rev 5Phos/aaagcacacgccccgg V182I-Fwd 5Phos/ATTacctgcgatgattataacgaagaaac V182I-Rev 5Phos/cagggtcaactgtttatctttgcc
eSrtA(4S-9) Primer Sequences (SEQ ID NOs: 68-73 from left to right and then top to bottom, respectively):
(135) TABLE-US-00005 V104A-Fwd 5Phos/GCGgaagaaaacgaaagcctggatgatc V104A-Rev 5Phos/aaagcacacaccacgatccag T118A-Fwd 5Phos/GCGggccataccgcgattgatcg T118A-Rev 5Phos/aatgctaatgttctgatcatccaggc V182I-Fwd 5Phos/ATTacctgcgatgattataactttgaaac V182I-Rev 5Phos/cagggtcagctgtttatctttgcc
Preparative-Scale Biotinylation of Fetuin A
(136) As in analytical labeling of Fetuin A, 1 mL of normal human plasma was combined with 10 L 1M CaCl2, 10 L of 0.1M GGGK(Biotin) (SEQ ID NO: 22), and 10 L of 100 M eSrtA(4S-9), then incubated at room temperature for 2 hours. 100 L of pre-equilibrated Ni-NTA resin slurry and 12.5 L 0.4M H2N-LPESGG (SEQ ID NO: 38) peptide was added to the mixture to act as a competitive inhibitor for thioester formation. The mixture was incubated at room temperature with shaking for 15 minutes, then filtered through a 0.2 m spin filter before dilution to 10 mL final volume in PBS+1 mM EDTA+100 M H2N-LPESGG (SEQ ID NO: 38) (PBS-EL). The solution was concentrated using a 10 kDa molecular weight cutoff spin concentrator for 20 minutes at 3500g and a final volume of <1 mL. This sample was diluted with PBS-EL to 10 mL final volume, re-concentrated, and re-diluted in a total of six wash cycles to give an expected small molecule biotin concentration of <1 nM. This concentrated mixture was then incubated with 200 L, of pre-equilibrated Invitrogen MyOne Streptavidin C1 Dynabeads with shaking for 30 minutes before magnetic separation and washing three times with PBS+0.1% Tween-20. The beads were then resuspended in 100 L SDS-PAGE loading buffer with 100 M free biotin and incubated at 95 C. for 15 minutes. A 15-L aliquot was then run on a 4-12% Bis-Tris PAGE gel and visualized by staining with coomassie blue. The 47 kDa band was excised with a clean razor. This sample was subjected to proteolytic digestion and analyzed by microcapillary reverse-phase HPLC nano-electrospray tandem mass spectrometry (LC/MS/MS) on a Thermo LTQOrbitrap mass spectrometer by the Harvard Mass Spectrometry and Proteomics Resource Laboratory, FAS Center for Systems Biology.
(137) Enzyme Specificity Assay
(138) Assays to determine eSrtA, eSrtA(2A-9) and eSrtA(4S-9) specificity were performed by preparing 10 M stocks of Abz-LXEXGK(Dnp) (SEQ ID NO: 75) peptides in 300 mM Tris pH 7.5, 150 mM NaCl, 5 mM CaCl2, 5% v/v DMSO, and 100 mM Gly-Gly-Gly-COOH (GGG). Reactions were performed by adding eSrtA(2A-9) to a final concentration of 450 nM, eSrtA(4S-9) to a final concentration of 115 nM or eSrtA to a final concentration of 47.5 nM and a final volume of 100 L, then incubating at 22.5 C. for 15 minutes. Reactions were quenched by the addition of 0.2 volumes of 5 M HCl, then were injected onto an analytical reverse-phase Eclipse XDB-C18 HPLC column (4.6150 mm, 5 m, Agilent Technologies) and chromatographed using a gradient of 10 to 65% acetonitrile with 0.1% TFA in 0.1% aqueous TFA over 13 minutes. Retention times under these conditions for the Abz-LPETGK(Dnp)-CONH2 (SEQ ID NO: 19) substrate, the released GKDnp peptide, and the Abz-LPETGGG-COOH (SEQ ID NO: 20) product were 12.8, 10.4, and 9.1 min, respectively, while the remaining Abz-LXEXGK(Dnp)-CONH2 (SEQ ID NO: 74) substrates varied in retention time between 9 and 13 minutes. To calculate the percent conversion, the ratio of the integrated areas of the GK(Dnp)-CONH2 and Abz-LPETGK(Dnp)-CONH2 (SEQ ID NO: 19) peptide Abs355 peaks were compared directly. Subsequent determination of k.sub.cat/K.sub.m and K.sub.H were performed as described for in vitro SrtA characterization.
(139) Differential Scanning Fluorimetry for Thermal Melting Curves
(140) To determine thermal stability of eSrtA, eSrtA(2A-9), eSrtA(4S-9), and variants thereof, each protein was freshly expressed and purified, then diluted to 40 M in 100 mM Tris pH 7.5, 500 mM NaCl. Differential scanning fluorimetry was performed using the Life Technologies Protein Thermal Shift Dye kit according to manufacturers' instructions. Thermal scanning was performed on Biorad CFX96-Real Time PCR (25 C. to 99 C., 0.2 C./2s increments). To calculate Tm, fluorescence intensity was fit to the Boltzmann equation using Microsoft Excel using the Solver add-in. Melting curves were plotted with best-fit fluorescence intensities that were normalized to maximum fluorescence intensity.
(141) GGG-Diblock Functionalization
(142) GGG-functionalized recombinant amphiphilic diblock polypeptide (Diblock), GGG-Diblock, was used as a substrate to test the orthogonality of the two evolved sortases in solution. GGG-Diblock based on elastin-mimetic polypeptide sequences was prepared as previously described (Kim et al. Self-Assembly of Thermally Responsive Amphiphilic Diblock Copolypeptides into Spherical Micellar Nanoparticles. Angewandte Chemie International Edition 49, 4257-4260, doi:10.1002/anie.201001356 (2010)) with slight changes in sequence by genetically incorporating an N-terminal triglycine motif. A complete amine acid sequence is GGG-VPGEG-[(VPGVG)(VPGEG)(VPGVG)(VPGEG)(VPGVG)].sub.10-CCCCGG-[(IPGVG).sub.2VPGYG(IPGVG).sub.2].sub.15-VPGYG (SEQ ID NO: 75). In a 0.6 mL Eppendorf tube, GGG-functionalized Diblock (10 M) was mixed with fluorophore-peptide (0.1 M), evolved sortase (15 M) and CaCl2 (100 mM), then reacted for 1 hour at room temperature. Samples with different combinations of fluorophore-peptide conjugates (Alexa Fluor 488-LAETG (SEQ ID NO: 5) or Alexa Fluor 647-LPESG (SEQ ID NO: 3)) and evolved sortases (eSrtA(2A-9) or eSrtA(4S-9)) were prepared to evaluate potential cross-reactivity. To analyze the reaction products, 4.7 L of 4SDS-PAGE loading buffer was added to 14.1 L of reaction mixture, incubated at 95 C. for 3 minutes, and electrophoresed on a 15% Tris-HCl Precast gel (BioRad) at 150 V for 60 minutes. After electrophoresis, fluorescent images were taken without any staining using GE Typhoon FLA 7000 Gel Scanner at Wyss Institute at resolution=25 m. After scanning, the gel was stained with coomasie blue to visualize protein bands containing GGG-diblock.
Example 1: Evolved Sortases Having Altered Substrate Specificity
(143) Sortase A (SrtA) from Staphylococcus aureus was evolved (mutated) to recognize and catalyze transpeptidation of substrates having altered sortase recognition motifs as described previously. See, e.g., Liu et al., U.S. patent application Ser. No. 13/922,812, filed Jun. 20, 2013, and Chen et al., PNAS, 108(28):11399-404 (2011), the entire contents of each are incorporated herein by reference. SrtA was evolved to recognize substrates having the altered sortase recognition motifs of LAETG (SEQ ID NO: 5) or LPESG (SEQ ID NO: 3), as compared to the canonical or wild type motif of LPETG (SEQ ID NO: 4). This process yielded 6 mutants recognizing the motif LAETG (SEQ ID NO: 5)(2Ar3, 2Ar3.5, 2Ar4, 2Ar5, 2Ar6a, and 2Ar6b;
(144) As depicted in
(145) As depicted in
Example 2: Evolved Sortases with Altered Substrate Specificity can be Used to Modify Proteins at Either or Both the N- and C-Terminal
(146) In order to test the utility of orthogonal Sortases for the construction of dual N- and C-terminally labeled material, GGG-FGF2/21-LPESG-HHHHHH (SEQ ID NO: 27) constructs were tested for SrtA reactivity. In brief, 10 uM of GGG-FGF-LPESG (SEQ ID NO: 3) construct was incubated for 1 hour in 100 mM Tris pH 7.5, 500 mM NaCl, 5 mM CaCl2 with one of 100 uM GGG-Biotin or 100 uM Biotin-LPESG and 5 uM SrtA. These reactions were quenched by the addition of 0.1 eq of SDS loading buffer, boiled, and then subjected to western blot analysis for the presence of Biotin and His5 moieties. In each case, biotinylation signal was observed in cognate pairs (e.g., SrtALAETG (SEQ ID NO: 5)/GGG-Biotin or SrtALPESG (SEQ ID NO: 3)/Biotin-LPESG (SEQ ID NO: 3)) but only in limited form in non-cognate pairs (
Example 3: Evolved Sortases can be Used for Specific Modification of Tissues
(147) Tissue staining was performed as described previously, with biotin signal false colored in red and DAPI stain false colored in blue. In each case, total integrated biotin signal was computed for both negative control and SrtA+ samples, then background subtracted and used to calculate the total fluorescence enrichment shown in
Example 4: Kinetics Data for Evolved Sortases
(148) TABLE-US-00006 TABLE 1 Kinetics data for evolved sortases. Substrate/Target wt/wt 5mut/wt 5mut/2A 2A.3.5/2A 2A.3.5/wt 2A.4/2A Mutations P94R D160N P94R D160N 5mut + K162R 5mut + K162R 5mut + A104H D165A K190E D165A K190E V168I I182F V168I I182F K162R V168I K196T K196T I182V kcat, 10 mM GGG 1.220694008 5.621882686 1.315536327 1.248895247 1.586795141 1.900782516 (Hz) Sdev, kcat, 10 mM 0.206127706 0.050996956 0.262907524 0.021650744 0.008262362 0.017265216 GGG (Hz) Km, LxExG (uM) 5180.865735 207.8069987 6156.781755 7162.52724 4109.913889 3251.169918 Sdev, Km, LxExG 943.4564337 27.66474456 874.4527319 513.1042324 104.44959 113.3307861 (uM) kcat/Km (Hz/M) 235.6158353 27053.38473 220.6162944 174.7051787 386.2215124 585.0234649 Sdev, kcat/Km 58.51434457 3609.890006 75.67625296 9.492630229 7.805955098 16.69296789 (Hz/M) specificity 122.6264126 2.21070443 73.04708671 (relative cat. Efficiency, fold) Sdev, specificity 45.13405627 0.128159767 3.589253382 (fold) kHydrolysis (Hz) 0.016574713 0.055463406 Sdev, kHydrolysis 0.008891304 0.040676362 (Hz) kcat, 1 mM LxExG 0.17308818 7.245083652 (Hz) Sdev, kcat, 1 mM 0.068138026 0.196190524 LxExG (Hz) Km, GGG (uM) 290.6170509 2987.626804 Sdev, Km, GGG 221.9726779 80.92902606 (uM) Khydrolysis (uM) 28.37924502 22.59305475 Sdev, Khydrolysis 21.646962 19.8961213 (uM) kL/KL/KH 8.30E+06 1.20E+09 Substrate/Target 2A.4/wt 2A.5/2A 2A.5/wt 2A.6A/2A 2A.6A/wt 2A.6B/2A Mutations 5mut + A104H 5mut + R99H 5mut + R99H 5mut + A104H 5mut + A104H 5mut + R99K K162R V168I A104H K138I A104H K138I K138V K162R K138V K162R A104H K138V I182V K162R I182V K162R I182V I182V I182V D160K* K162R I182V kcat, 10 mM GGG 0.093555041 1.409999086 0.137531365 2.469983633 0.15719454 0.69767657 (Hz) Sdev, kcat, 10 mM 0.009667427 0.055318463 0.005753656 0.148040476 0.013824772 0.019749868 GGG (Hz) Km, LxExG (uM) 11669.27262 433.3119304 4897.457635 608.3313756 3289.020619 309.9866552 Sdev, Km, LxExG 896.6218943 7.000387377 392.6284437 100.775363 298.3210472 28.84077154 (uM) kcat/Km (Hz/M) 8.008854169 3255.914366 28.14079796 4128.180146 47.80226835 2266.706448 Sdev, kcat/Km 0.296497092 179.204205 1.096470124 646.4782722 0.58178654 261.6475808 (Hz/M) specificity 115.7008543 86.35950319 (relative cat. Efficiency, fold) Sdev, specificity 11.30238355 5.406626636 (fold) kHydrolysis (Hz) 0.324012523 0.225234539 0.010481967 Sdev, 0.025489666 0.078893389 0.002163477 kHydrolysis (Hz) kcat, 1 mM LxExG 1.492620267 1.71404659 0.535930039 (Hz) Sdev, kcat, 1 mM 0.07081996 0.069056717 0.031080138 LxExG (Hz) Km, GGG (uM) 2806.036691 1039.788533 642.2785789 Sdev, Km, GGG 366.9256799 237.2398425 25.59263171 (uM) Khydrolysis (uM) 615.8786441 135.7509813 12.49068866 Sdev, 140.4338096 60.22602109 2.764212493 Khydrolysis (uM) kL/KL/KH 5.29E+06 3.04E+07 1.81E+08 Substrate/Target 2A.6B/wt 2A.6C/2A 2A.6C/wt 5mut/4S 4S.3.5/4S 4S.3.5/wt Mutations 5mut + R99K 5mut + A104H 5mut + P94R D160N 5mut A104T 5mut A104T A104H K138P K152I A104H D165A K190E A118T I182V A118T I182V K138V D160K* K138P K152I K196T D160K* K162R I182V D160K* K162R I182V K162R I182V kcat, 10 mM GGG 0.812860117 0.07675539 1.739673591 1.118147686 (Hz) Sdev, kcat, 10 mM 0.09198838 0.002728785 0.174911912 0.164087961 GGG (Hz) Km, LxExG (uM) 343.4888498 1983.065489 671.940841 7310.434447 Sdev, Km, LxExG 24.03918885 77.67622015 167.1750208 1308.709174 (uM) kcat/Km (Hz/M) 2384.915833 38.70920459 2654.656128 153.8703055 Sdev, kcat/Km 428.6545792 0.191909734 436.5540965 12.72468908 (Hz/M) specificity (relative 61.61107824 10.19091868 cat. Efficiency, fold) Sdev, specificity 11.07792379 2.158175931 (fold) kHydrolysis (Hz) 0.010481967 Sdev, kHydrolysis 0.002163477 (Hz) kcat, 1 mM LxExG 0.535930039 (Hz) Sdev, kcat, 1 mM 0.031080138 8.841950988 LxExG (Hz) Km, GGG (uM) 642.2785789 Sdev, Km, GGG 25.59263171 (uM) Khydrolysis (uM) 12.49068866 Sdev, Khydrolysis 2.764212493 (uM) kL/KL/KH 1.91E+08 Substrate/Target 4S.4/4S 4S.4/wt 4S.5/4S 4S.5/wt 4S.6A/4S 4S.6A/wt Mutations 5mut + A104V 5mut + A104V 5mut + N98D 5mut + N98D 5mut + N98D 5mut + N98D A118T F122S A118T F122S A104V A118T A104V A118T A104V A118S A104V A118S I182V I182V F122A K134R F122A K134R F122A K134G F122A K134G I182V I182V I182V E189V I182V E189V kcat, 10 mM GGG 0.726925959 0.095714145 0.387725911 0.006148609 0.953745987 1.102146085 (Hz) Sdev, kcat, 10 mM 0.040738822 0.006560006 0.023554298 0.000608698 0.095078853 0.045369351 GGG (Hz) Km, LxExG (uM) 1408.091941 338.7530289 75.99463534 12.18687654 102.3903263 1294.3279 Sdev, Km, LxExG 103.4411288 35.89713184 8.975668999 1.361443909 10.64783321 110.7342418 (uM) kcat/Km (Hz/M) 516.9021989 283.4849351 5129.750723 513.0401911 9394.140707 854.1552296 Sdev, kcat/Km 19.06475492 15.31339113 392.5617968 114.3429095 1448.405056 53.20038819 (Hz/M) specificity 1.823385072 9.998730727 10.99816565 (relative cat. Efficiency, fold) Sdev, specificity 0.24057593 0.160364958 12.6411151 (fold) kHydrolysis (Hz) 0.009550657 0.066957275 Sdev, kHydrolysis 0.000364915 0.045974206 (Hz) kcat, 1 mM LxExG 1.34223901 1.930863111 (Hz) Sdev, kcat, 1 mM 0.328144639 0.13952882 LxExG (Hz) Km,GGG (uM) 26698.41922 4946.346135 Sdev, Km, GGG 6134.780703 1121.104516 (uM) Khydrolysis (uM) 190.7665753 179.292082 Sdev, Khydrolysis 17.86055357 148.1163037 (uM) kL/KL/KH 2.69E+07 5.24E+07 Substrate/Target 4S.6B/4S 4S.6B/wt 4S.6C/4S 4S.6C/wt Mutations 5mut + N98D 5mut + N98D 5mut + N98D 5mut + N98D A104V A118S A104V A118S A104V A118T A104V A118T F122A K134P F122A K134P F122A K134R F122A K134R I182V E189P I182V E189P I182V E189F I182V E189F kcat, 10 mM GGG (Hz) 1.241539966 1.080724703 0.665737178 0.003663126 Sdev, kcat, 10 mM 0.108271856 0.101425203 0.16708905 0.000119561 GGG (Hz) Km, LxExG (uM) 311.9338134 237.5991947 156.478353 16.05552448 Sdev, Km, LxExG (uM) 94.98617736 17.47052549 28.44546328 3.73862534 kcat/Km (Hz/M) 4139.211117 4547.346117 4349.070663 234.8365865 Sdev, kcat/Km (Hz/M) 788.9272214 219.0365622 1447.408578 43.35556701 specificity (relative 0.91024765 18.51956174 cat. Efficiency, fold) Sdev, specificity 0.761697464 0.102605403 (fold) kHydrolysis (Hz) 0.016704884 0.013989042 Sdev, kHydrolysis 0.001313081 0.002355155 (Hz) kcat, 1 mM LxExG (Hz) 2.131849488 1.502291868 Sdev, kcat, 1 mM 0.114779707 0.02193525 LxExG (Hz) Km, GGG (uM) 15730.32228 35983.54434 Sdev, Km,GGG (uM) 2130.787148 1480.295766 Khydrolysis (uM) 122.32836 334.1419054 Sdev, Khydrolysis 4.050583082 63.1306735 (uM) kL/KL/KH 3.38E+07 1.30E+07
Example 5: Reprogramming the Specificity of Sortase Enzymes
(149) S. aureus sortase A catalyzes the transpeptidation of an LPXTG (SEQ ID NO: 2) peptide acceptor and a glycine-linked peptide donor and has proven to be a powerful tool for site-specific protein modification. The substrate specificity of sortase A is stringent, limiting its broader utility. Here we report the laboratory evolution of two orthogonal sortase A variants that recognize each of two altered substrates, LAXTG (SEQ ID NO: 9) and LPXSG (SEQ ID NO: 8), with high activity and specificity. Following nine rounds of yeast display screening integrated with negative selection, the evolved sortases exhibit specificity changes of up to 51,000-fold relative to the starting sortase without substantial loss of catalytic activity, and with up to 24-fold specificity for their target substrates relative to their next most active peptide substrate. The specificities of these altered sortases are sufficiently orthogonal to enable the simultaneous conjugation of multiple peptide substrates to their respective targets in a single solution. We demonstrated the utility of these evolved sortases by using them to effect the site-specific modification of endogenous fetuin A in human plasma, the synthesis of tandem fluorophore-protein-PEG conjugates for two therapeutically relevant fibroblast growth factor proteins (FGF1 and FGF2), and the orthogonal conjugation of fluorescent peptides onto surfaces.
(150) The modification of proteins has proven to be crucial for many research and industrial applications. The bacterial transpeptidase S. aureus Sortase A (SrtA) is a powerful tool for conjugating proteins to a wide variety of molecules, but is limited to those proteins containing the five amino acid LPXTG (SEQ ID NO: 2) sorting motif. Here we present a system for the directed evolution of reprogrammed SrtA variants that accept proteins with altered sorting motifs. We used this system to evolve two families of orthogonal sortases that recognize LAXTG (SEQ ID NO: 9) and LPXSG (SEQ ID NO: 8) motifs. These evolved sortases enabled the synthesis of triblock fluorophore-protein-PEG conjugates, the covalent and orthogonal functionalization of multiple proteins onto surfaces, and the manipulation of endogenous human proteins lacking a native LPXTG (SEQ ID NO: 2) motif.
(151) The laboratory modification of proteins enables applications including the manipulation of protein pharmacokinetics (37), the study of protein biochemistry (38), the immobilization of proteins (39), and the synthesis of protein-protein fusions that cannot be expressed in cells (40). An attractive approach for the synthesis of protein conjugates attaches molecules site-specifically to proteins using epitope-specific enzymes. Such a strategy can overcome the challenges of bioorthogonality and chemoselectivity through the careful choice of enzyme and epitope. Techniques to implement this approach, however, are commonly limited by the requirement of cumbersome and poorly-tolerated fusion epitopes, or by rigidly defined enzyme substrate specificity.
(152) The bacterial transpeptidase S. aureus sortase A (SrtA) mediates the anchoring of proteins to the bacterial cell wall and has been widely used in bioconjugate synthesis (41). Wild-type SrtA binds a small, five-amino acid sorting motif (Leu-Pro-X-Thr-Gly, LPXTG, SEQ ID NO: 2, where X=any amino acid) and cleaves the scissile Thr-Gly peptide bond via a cysteine protease-like mechanism, resulting in loss of the C-terminal glycine to yield a thioacyl intermediate. This intermediate reacts with an N-terminal Gly-Gly-Gly motif to generate a -LPXTGGG-product (SEQ ID NO: 76)(
(153) The utility of wild-type SrtA has been limited by two main factors. First, its poor catalytic activity reduces reaction yields and can lead to stable thioacyl intermediates, limiting the use of wild-type SrtA to small-scale bioconjugate synthesis in which superstoichiometric enzyme loadings and long timescales are tolerated. To overcome this limitation, we recently developed a system for the evolution of bond-forming enzymes based on yeast display (
(154) A second limitation of both SrtA (46) and eSrtA is their requirement for substrates containing LPXTG (SEQ ID NO: 2)(
(155) In this work we developed and applied a modified bond-forming enzyme screening system to enable the laboratory evolution of SrtA variants with dramatically altered, rather than broadened, substrate specificity. Over nine rounds of mutagenesis and screening with concomitant refinement of our library design and screening strategy, we evolved reprogrammed eSrtA variants that recognize either LPXSG (SEQ ID NO: 8) or LAXTG (SEQ ID NO: 9) with up to a 51,000-fold change in specificity and minimal loss of activity relative to eSrtA. We used one of the altered sortases to achieve the post-translational modification of endogenous fetuin A in human plasma, which cannot be efficiently modified by eSrtA because it lacks an LPXTG (SEQ ID NO: 2) motif. In addition, we used the reprogrammed sortases to mediate the rapid synthesis of doubly modified fluorophore-protein-PEG conjugates. Finally, we used the evolved sortases to immobilize orthogonal fluorophore-linked peptides onto GGG-conjugated surfaces. Collectively, these findings establish a facile approach for generating sortase enzymes with tailor-made substrate specificities and greatly expand the number of highly active, orthogonal sortase enzymes available for protein conjugation applications.
(156) Table 5 shows rate constants for two exemplary evolved eSrtA derivatives 2A-9 (also referred to as eSrtA(2A-9)) and 4S-9 (also referred to as eSrtA(4S-9)). The original pentamutant comprising the mutations P94R, D160N, D165A, K190E, and K196T is referred to as eSrtA. Sortase variants herein were evolved using eSrtA as the parent enzyme in the evolution experiments. In some cases, an evolved sortase comprises a N160K mutation instead of a D160N or a T196S mutation instead of a K196T. Parameters are reported with their standard deviations as determined from three technical replicates. k.sub.cat, K.sub.m and k.sub.cat/K.sub.m parameters were determined at 100 mM GGG concentration, while K.sub.m,GGG and K.sub.H parameters were determined at 1 mM concentration of the listed substrate.
(157) TABLE-US-00007 TABLE 5 Rate constants for evolved eSrtA derivatives 2A-9 (also referred to as eSrtA(2A-9)) and 4S-9 (also referred to as eSrtA(4S-9)). SrtA is the wild-type sortase. (SEQ ID NOs: 85-101 from top to bottom) Enz Sub K.sub.cat (Hz) K.sub.m (M) k.sub.cat/K.sub.m (M.sup.1s.sup.1) K.sub.m,GGG (M) K.sub.H (M) SrtA LPETG 1.5 0.2 7600 500 200 30 211 8 14.3 0.8 eSrtA LPETG 5.4 0.4 230 20 23000 3000 1617 13 32.8 2.4 eSrtA LAETG 1.31 0.26 6066 870 223 77 807 56 26.5 0.6 eSrtA LSETG 0.102 0.001 533 24 192 9 337 16 37.4 3.4 eSrtA LPEAG 0.74 0.04 83.4 0.6 8900 500 48400 3100 599 41 eSrtA LPECG 3.5 0.3 72.5 5.6 48800 8100 57000 33000 620 150 eSrtA LPESG 1.46 0.13 318 58 4650 466 22000 900 76 19 eSrtA(2A-9) LPETG 0.0209 0.004 1267 62 16.5 0.4 585 3 27.1 0.1 eSrtA(2A-9) LAETG 2.23 0.02 265 9 8421 311 1480 190 33 4 eSrtA(2A-9) LRETG 0.25 0.02 1072 63 233 34 628 11 47.5 1.3 eSrtA(2A-9) LSETG 0.115 0.007 331 67 355 54 1270 200 26.0 3.4 eSrtA(2A-9) LPESG 0.0066 0.0006 4800 1200 1.42 0.26 eSrtA(4S-9) LPETG 0.047 0.006 64.7 4.2 720 41 98000 13000 295 19 eSrtA(4S-9) LAETG 0.0078 0.0006 387 49 20.2 0.9 eSrtA(4S-9) LPEAG 1.90 0.01 154 3 12300 200 41000 4000 193 30 eSrtA(4S-9) LPECG 5.0 0.6 69 13 74000 5300 55000 1400 120 23 eSrtA(4S-9) LPESG 2.05 0.15 113 12 18160 1950 69000 12000 174 32
A Competitive Inhibition Strategy for Sortase Evolution.
(158) To enable the evolution of eSrtA variants with altered substrate specificity, we modified our previously described yeast display screen (45) with the addition of a negative selection against recognition of off-target substrates (
(159) To test the ability of cognate LPETG (SEQ ID NO: 4) substrate (K.sub.m=0.2 mM) to compete with a candidate LAETG (SEQ ID NO: 5) target (K.sub.m=6.1 mM) in our system, we used S. cerevisiae displaying eSrtA and GGG on the cell surface as previously described (9). We incubated these cells with 10 M biotin-LAETG (SEQ ID NO: 5) and a range of 100 nM to 1 mM non-biotinylated LPETG (SEQ ID NO: 4) for 1 hour (
(160) Initial Evolution of eSrtA Variants with Altered Substrate Preference.
(161) Because binding-pocket geometries of SrtA in previously reported structures are diverse (50-52), we used broad, unbiased mutagenesis to generate initial eSrtA diversity. We randomized the SrtA gene (441 bp) at a 2% mutation level using chemical mutagenesis, transformed the resulting gene pool into yeast to generate libraries of 10.sup.7 to 10.sup.8 eSrtA variants, and screened the resulting libraries after incubation with either biotin-LAETG (SEQ ID NO: 5) or biotin-LPESG (SEQ ID NO: 3) in the presence of various concentrations of LPETG (SEQ ID NO: 4) as a competitive inhibitor (Table 4). Starting eSrtA exhibits a 103-fold preference for LPETG (SEQ ID NO: 4) over LAETG (SEQ ID NO: 5), and a 5-fold preference for LPETG (SEQ ID NO: 4) over LPESG (SEQ ID NO: 3)(
(162) Table 4 shows Evolutionary history of eSrtA(2A-9) and eSrtA(4S-9) enzymes. In each case, libraries were iteratively selected against increasing concentrations of the off-target substrate LPETGG (SEQ ID NO: 36) in the presence of decreasing concentrations of biotinylated LAETGG (SEQ ID NO: 35) or LPESGG (SEQ ID NO: 38).
(163) TABLE-US-00008 # Sorts Final Substrate Final Off-Target Final Library per Concentration Substrate Incubation Final Substrate Round Size round (M) Concentration (M) Time (min) Library Size LAETG 1 1.0 10.sup.8 4 10 0.1 60 1 10.sup.5 LAETG 2 4.6 10.sup.7 4 10 10 60 1 10.sup.5 LAETG 3 6.8 10.sup.7 4 1 10 60 1 10.sup.5 LAETG 4 4.8 10.sup.7 6 0.1 100 60 8.2 10.sup.4 LAETG 5 3.8 10.sup.7 5 0.01 100 60 7 10.sup.4 LAETG 6 5.4 10.sup.7 7 0.01 1000 60 7.2 10.sup.4 LAETG 7 4.1 10.sup.7 3 0.01 1000 60 3.2 10.sup.4 LAETG 8 2.6 10.sup.7 4 0.01 1000 5 5 10.sup.4 LAETG 9 8.9 10.sup.7 5 0.01 1000 5 4 10.sup.4 LPESG 1 1.0 10.sup.8 4 1 0.1 60 1 10.sup.5 LPESG 2 6.0 10.sup.7 4 0.1 10 60 1 10.sup.5 LPESG 3 4.7 10.sup.7 4 0.01 100 60 1 10.sup.5 LPESG 4 6.1 10.sup.7 6 0.01 100 60 9 10.sup.4 LPESG 5 3.2 10.sup.7 6 0.01 1000 60 3 10.sup.4 LPESG 6 5.1 10.sup.7 7 0.01 1000 60 7 10.sup.4 LPESG 7 4.8 10.sup.7 3 0.01 1000 60 1.6 10.sup.4 LPESG 8 2.8 10.sup.7 4 0.01 1000 5 1.5 10.sup.6 LPESG 9 1.0 10.sup.8 5 0.006 3000 5 1.2 10.sup.5
Table 4 shows Evolutionary history of eSrtA(2A-9) and eSrtA(4S-9) enzymes. LAETG: SEQ ID NO: 5; LPESG: SEQ ID NO: 4.
(164) Three rounds of whole-gene mutagenesis and screening yielded two converged clones, eSrtA(2A-3) and eSrtA(45-3), each of which contained between eight and ten coding mutations relative to eSrtA (
(165) Secondary Evolution of eSrtA Variants with Altered Substrate Specificity.
(166) Next we generated site-saturation libraries based on the eSrtA(2A-3.5) and eSrtA(4S-3.5) minimal mutants. Using degenerate NNK codons, we randomized residues 104, 168, and 182 in eSrtA(2A-3.5), and residues 104, 118, and 182 in eSrtA(4S-3.5). Additionally, we used PCR mutagenesis to further diversify these libraries in an untargeted manner at a mutation rate of 1% per residue. Screening of these libraries against their target substrates in the presence of ten-fold higher concentrations of non-biotinylated LPETG (SEQ ID NO: 4) yielded the round 4 consensus variants eSrtA(2A-4) and eSrtA(45-4), each of which acquired novel mutations (
(167) Repeating this process, we applied NNK mutagenesis to the most frequently mutated residues among clones emerging from round 4 (positions 162, 168, and 182 in eSrtA(2A-4), and positions 118, 122, and 182 in eSrtA(45-4)), and also mutated the rest of the eSrtA gene at a 1% frequency. Screening of the resulting libraries yielded consensus variants eSrtA(2A-5) and eSrtA(45-5), each of which included a mixture of mutations at targeted and untargeted residues (
(168) Finally, we repeated this approach of saturation mutagenesis on each of the newly discovered mutations, as well as the original five mutations in eSrtA, which are P94R, D160N, D165A, K190E, and K196T. Starting from eSrtA(2A-5), we combined five libraries in which positions 99 and 138 were randomized, in addition to residue 160, 165, 189, 190, or 196. Similarly, starting from eSrtA(45-5), we randomized position 132, in addition to residue 160, 165, 189, 190, or 196, before combining the resulting five libraries. Screening provided consensus variants eSrtA(2A-6) and eSrtA(45-6). These variants exhibited only marginal improvements in catalytic activity and specificity relative to round 5 clones (
(169) Given that substrate specificity and catalytic activity were comparable among clones emerging from rounds 5 and 6, we hypothesized that the advantage of eSrtA(2A-6) and eSrtA(45-6) over their ancestors could arise from reduced substrate hydrolysis. To decouple hydrolysis from overall enzymatic efficiency, we measured the concentration of GGG at which the rates of acyl-enzyme hydrolysis and transpeptidation are equal, which we define as the parameter K.sub.H. We found that eSrtA(2A-6) (K.sub.H=14963 M) possessed significantly improved hydrolytic stability when compared with eSrtA(2A-5) (K.sub.H=731235) and that eSrtA(45-6) (K.sub.H=11610 M) was also improved relative to eSrtA(45-5) (K.sub.H=19016 M).
(170) Taken together, these results suggest that the use of whole-gene mutagenesis to identify target loci for targeted mutagenesis provides access to eSrtA variants with altered substrate specificities. Despite the strong gains observed in rounds 4 and 5, however, we observed no significant activity gains in round 6, suggesting that these evolved enzymes were in a local fitness maximum.
(171) Evolving Highly Active eSrtA Mutants with Altered Specificities.
(172) In an effort to escape putative fitness maxima, we partially randomized all of the residues mutated among clones in rounds 4, 5, and 6. For each target site, we created degenerate codon libraries with a 27% mutagenesis rate at each nucleotide of each codon observed to change from our eSrtA starting scaffold. We applied this scheme to residues 94, 98, 99, 104, 160, 162, 165, 182, 189, 190, and 196 in both eSrtA(2A-6) and eSrtA(45-6) and further applied broad spectrum mutagenesis to each library at a level of approximately 1%. Beginning from this highly mutated starting material, we screened each successive library with gradually increasing stringency by decreasing incubation times before sorting to only 5 minutes in round 7 (Table 4). The round 7 survivors were randomly mutagenized and rescreened at 5-minute incubation times, decreased concentrations of biotinylated substrate, and/or increased concentrations of non-biotinylated LPETG (SEQ ID NO: 4), yielding libraries 8 and 9 (Table 4). These libraries converged on clones eSrtA(2A-9) and eSrtA(4S-9), each possessing four or five mutations relative to their round 6 counterparts. These mutants were highly active on their target substrates and minimally active on LPETG (SEQ ID NO: 4). The overall changes in substrate specificity of eSrtA(2A-9) and eSrtA(4S-9) relative to eSrtA are 51,000- and 120-fold, respectively (
(173) Clone eSrtA(4S-9) exhibited strong preference for LPESG (SEQ ID NO: 3) over LPETG (SEQ ID NO: 4)(25-fold) that was greater than eSrtA's opposite starting preference for LPETG (SEQ ID NO: 4) over LPESG (SEQ ID NO: 3)(5-fold), and showed negligible activity on LAETG (SEQ ID NO: 5). We hypothesized that its specificity for LPESG (SEQ ID NO: 3) was in part caused by the eSrtA(4S-9)(LPES; SEQ ID NO: 18) intermediate coupling more efficiently to GGG compared to the eSrtA(4S-9)(LPET) intermediate. In order to test this possibility, we measured K.sub.H of eSrtA(4S-9) for LPETG (SEQ ID NO: 4) and LPESG (SEQ ID NO: 3), and observed that K.sub.H,LPETG (29518 M) was nearly twice that of K.sub.H,LPESG (17432 M). These results illustrate that eSrtA(4S-9) achieves its specificity via a combination of selectively binding LPESG (SEQ ID NO: 3) over LPETG (SEQ ID NO: 4), as well as reduced stability of the mischarged SrtA(LPET; SEQ ID NO: 18) intermediate. Collectively, these features led to an approximately 40-fold difference in transpeptidation of LPESG (SEQ ID NO: 3) over LPETG (SEQ ID NO: 4) by eSrtA(4S-9) (
(174) Similarly, eSrtA(2A-9) showed dramatically higher specificity than eSrtA for its target substrate, with a nearly 500-fold preference for LAETG (SEQ ID NO: 5) over LPETG (SEQ ID NO: 4) as compared to eSrtA's 103-fold opposite starting specificity for LPETG (SEQ ID NO: 4) over LAETG (SEQ ID NO: 5), and negligible activity on LPESG (SEQ ID NO: 3). Measuring K.sub.H,LPETG (18.73.3 M) of eSrtA(2A-9) revealed that eSrtA(2A-9) has substantially improved hydrolytic stability compared to that of eSrtA (K.sub.H,LPETG=32.82.4 M), and comparable to that of wild-type SrtA (K.sub.H,LPETG=14.30.8 M) (Table 2).
(175) Table 2 shows the kinetic parameters for wild-type SrtA, eSrtA, eSrtA(2A-9), and eSrtA(4S-9). In each case, rate constants were determined by measuring enzyme velocity at eight different substrate concentrations by HPLC assay, then fit using nonlinear regression to the Michaelis-Menten equation, yielding k.sub.cat and K.sub.m. K.sub.H was calculated by measuring enzyme velocity at 1 mM target substrate and eight different GGG concentrations by HPLC assay, then fitting the resulting curves using nonlinear regression to the modified Michaelis-Menten equation.
(176) TABLE-US-00009 TABLE 2 Kinetic parameters for wild-type SrtA, eSrtA, eSrtA(2A-9), and eSrtA(4S-9). Enz Sub k.sub.cat (Hz) K.sub.m (M) k.sub.cat/K.sub.m (M.sup.1s.sup.1) Rel. Activity K.sub.N (M) SrtA LPETG 1.5 0.2 7600 500 200 30 1 14.3 0.8 eSrtA LPETG 5.4 0.4 230 20 23000 3000 1 32.8 2.4 eSrtA LAETG 1.31 0.26 6070 870 223 77 103.3 26.5 0.6 eSrtA LPESG 1.46 0.13 318 58 4650 466 4.9 76 19 eSrtA(2A-9) LPETG 0.0209 0.0004 1267 62 16.5 0.4 510 27.1 0.1 eSrtA(2A-9) LAETG 2.23 0.02 265 9 8421 311 1 33 4 eSrtA(2A-9) LPESG 0.0066 0.0006 4800 1200 1.42 0.26 5943 eSrtA(4S-9) LPETG 0.047 0.006 64.7 4.2 720 41 25 295 19 eSrtA(4S-9) LAETG 0.0078 0.0006 387 49 20.2 0.9 898 eSrtA(4S-9) LPESG 2.05 0.15 113 12 18000 2000 1 174 32
(177) In order to test if eSrtA(2A-9) and eSrtA(4S-9) favor their respective targets over related peptides to a similar extent as that of wild-type SrtA (10) and eSrtA, we profiled the activity of eSrtA(2A-9), eSrtA(4S-9), and eSrtA on 20 variants of each of their respective peptide targets containing all possible amino acid substitutions at position 2 or position 4 (
(178) Next, we dissected key structure-activity relationships among evolved sortases by reverting first-shell active site residues of both eSrtA(2A-9) and eSrtA(4S-9) back to that of eSrtA and assaying their respective activities (
(179) Individually reverting the first-shell residues V104 and T118 in eSrtA(4S-9) resulted in promiscuous enzymes, lowering specificity for LPESG (SEQ ID NO: 3) over LPETG (SEQ ID NO: 4) by 14-fold for the V104A mutant and by 75-fold for the T118A mutant. Reversion of V182 in eSrtA(4S-9) preserved selectivity but decreased overall activity, consistent with the effect of this reversion in eSrtA(2A-9). Taken together, these results suggest that residues 104 and 118 both impact specificity at the fourth position of the sorting motif. In addition, thermal melt assays revealed that the eSrtA(2A-9) and eSrtA(4S-9) point mutants described above each possess modestly higher thermal stability than their respective eSrtA(2A-9) or eSrtA(4S-9) parental enzymes (
(180) Collectively, these results establish that eSrtA(2A-9) and eSrtA(4S-9) evolved highly altered but quite stringent specificity, at least in part modulated through a novel process involving the differential hydrolytic stability of their acyl-enzyme intermediates. Both evolved round 9 clones strongly prefer their new LAXTG (SEQ ID NO: 9) or LPXSG (SEQ ID NO: 8) targets over the canonical LPXTG (SEQ ID NO: 2) substrate, yet maintain comparable overall catalytic efficiency as that of eSrtA (
(181) eSrtA(4S-9) Modifies Endogenous Fetuin A in Human Plasma
(182) In light of the known activity of eSrtA in human serum (18) and the highly altered specificity of the reprogrammed sortases, we hypothesized that eSrtA(4S-9) could catalyze the site-selective modification of endogenous LPXSG (SEQ ID NO: 8) or LPXAG (SEQ ID NO: 82) motifs in the human proteome. Based on an initial survey of the Uniprot protein database (55), we identified 199 candidate proteins with LPXSG (SEQ ID NO: 8) or LPXAG (SEQ ID NO: 82) motifs known to exist in the human proteome. Cross-validation against the Plasma Proteome Database (56) identified 36 proteins known to be present at detectable concentrations in human plasma. Due to the frequent occlusion of such tags in their folded state, we speculated that only a small fraction of these 36 candidate proteins would be accessible by an enzyme.
(183) We tested the ability of eSrtA(4S-9) to label proteins in human plasma by co-incubating whole plasma with eSrtA(4S-9) in the presence of Gly-Gly-Gly-Lys(Biotin) in the presence or absence of 10 mM CaCl.sub.2. Immunoblot and biotin capture each identified a single transpeptidation product (
(184) These findings demonstrate the ability of reprogrammed sortase enzymes to conjugate substrates to endogenous human proteins without chemical or genetic intervention. The high activity level of eSrtA(4S-9) in the absence of supplemental calcium demonstrates that evolved eSrtA variants can modify endogenous proteins with no additional cofactors.
(185) Reprogrammed Sortases Enable the Facile Synthesis of Complex Bioconjugates.
(186) The multiple modification of a protein's N- and C-terminus using only a single SrtA enzyme is challenging due to competing reactions of protein oligomerization and circularization whenever a reactive C-terminal sorting peptide and N-terminal GGG are both present (
(187) We expressed recombinant FGF1 and FGF2 as SUMO-TEV-GGG-FGF-LPESG-His.sub.6 (SEQ ID NO: 27) constructs (where SUMO indicates a Small Ubiquitin-like MOdifier tag (65), and TEV indicates a Tobacco Etch Virus protease cleavage site). Due to the close proximity of the FGF N- and C-termini, transpeptidation attempts using an unprotected N-terminus, or using non-orthogonal enzymes, generated only circularized FGF (
(188) Table 3 shows the yields of FGF/GGG-PEG semisynthesis. 750-FGF-GGG-PEG conjugates were synthesized as described in main text, using intermediate purification by Ni-NTA filtration to remove SrtA catalyst and unreacted starting material, then concentrated using 10 kDa MWCO membranes to eliminate residual GGG-PEG and leaving groups. These conjugates were assayed for purity via denaturing gel electrophoresis, and for overall yield by BCA assay, shown above. FGF2 conjugates showed uniformly poorer yield than FGF1 conjugates, likely due to their lower starting purity than FGF1 (<50% for FGF2, as opposed to >90% for FGF1) and to their smaller reaction scale (0.5 mg versus 8 mg).
(189) TABLE-US-00010 TABLE 3 Yields of FGF/GGG-PEG semisynthesis. Starting Conjugate Protein C-Terminal Modifier Protein, mg Protein, mg Yield FGF1 GGG 8.08 1.48 31.0% FGF1 GGG-PEG.sub.10 kDa 8.08 1.25 16.7% FGF1 GGG-bis-PEG.sub.10 kDa 8.08 0.84 11.2% FGF1 GGG-4arm-PEG.sub.10 kDa 8.08 1.48 19.6% FGF1 GGG-PEG.sub.10 kDa-Biotin 8.08 0.84 11.1% FGF2 GGG 0.54 0.01 0.4% FGF2 GGG-PEG.sub.10 kDa 0.54 0.10 2.0% FGF2 GGG-bis-PEG.sub.10 kDa 0.54 0.10 1.8% FGF2 GGG-4arm-PEG.sub.10 kDa 0.54 0.10 1.9% FGF2 GGG-PEG.sub.10 kDa-Biotin 0.54 0.08 1.6%
(190) These results establish that eSrtA(2A-9) and eSrtA(4S-9) form an orthogonal protein conjugation enzyme pair, enabling the direct synthesis of complex bioconjugates at substantially improved scale and yield relative to previous methods (48). The facile and parallel synthesis of milligram quantities of dual PEG- and fluorophore-conjugated proteins of clinical interest may facilitate the high-throughput synthesis and screening of bioconjugates at scales relevant to preclinical studies.
(191) eSrtA(2A-9) and eSrtA(4S-9) Modify Material Surfaces Orthogonally and with High Activity.
(192) Encouraged by the effectiveness of eSrtA(2A-9) and eSrtA(4S-9) for protein semisynthesis, we tested their potential utility for functionalizing surface materials. Although previous methods of biofunctionalization have been successful in generating materials with improved biocompatibility (66, 67), these methods are only compatible with end-point immobilization of a single protein. Techniques for the orthogonal or multi-component immobilization of several proteins to a single material target could enable the synthesis of more sophisticated protein-linked materials.
(193) To test the ability of our evolved eSrtA variants to selectively modify their cognate substrates in complex mixtures, we measured their ability to modify GGG-functionalized amphiphilic diblock polypeptide using fluorophore-conjugated LAETG (SEQ ID NO: 5) or LPESG (SEQ ID NO: 3)(
(194) Kinetics Data from Sortase Variants Found in Example 5.
(195) TABLE-US-00011 Specificity (relative catalytic Substrate/ Avg k.sub.cat Sdev Avg Sdev Avg Sdev efficiency, Target (Hz) k.sub.cat K.sub.M (uM) K.sub.M (uM) kcat/K.sub.M k.sub.cat/K.sub.M fold) 2A-3/2A 1.117413 0.318567 1615.404 849.9625 881.2388 475.5349 2A-3/wt 2.653092 0.062306 2102.414 162.4465 1265.372 66.88818 1.4359011 2A-3.5/2A 1.13998 0.173449 6374.087 1307.152 180.3628 11.64569 2A-3.5/wt 1.524789 0.012445 3754.747 99.56149 406.2295 7.593709 2.2522914 2A-4/2A 1.987692 0.081364 3724.32 180.5807 533.8351 4.397007 55.106065 2A-4/wt 0.0501 0.001285 5177.212 151.5023 9.68741 0.532558 2A-6/2A 2.578821 0.075357 664.64 83.42178 3927.566 568.1306 82.39868 2A-6/wt 0.162153 0.011155 3402.008 232.9357 47.6654 0.704511 2A-9/2A 2.229669 0.019075 264.9965 8.966399 8420.766 310.651 509.8362776 2A-9/wt 0.020915 0.000396 1267.604 61.71959 16.51707 0.55393 Substrate/ Avg k.sub.cat Avg K.sub.M Sdev K.sub.M Avg k.sub.cat/ Sdev Target (Hz) Sdev k.sub.cat (uM) (uM) K.sub.M kcat/K.sub.M Specificity 4S-3/4S 2.543424 0.033133 3333.115 57.62426 763.1319 7.111243 244.976 4S-3/wt 0.007565 0.000171 3387.672 2584.606 3.115125 1.806966 4S-3.5/4S 5.779208 4.378486 38816.43 36402.39 193.0372 77.85634 1.45405 4S-3.5/wt 5.482302 4.774694 39299.79 35851.82 132.758 88.57179 4S-4/4S 0.757397 0.024013 1556.521 26.36027 486.5422 9.459991 2.22358 4S-4/wt 0.107323 0.005682 491.1604 35.58553 218.8102 8.523971 4S-5/4S 0.387727 0.023553 75.99485 8.97524 5129.744 392.5421 9.67204 4S-5/wt 0.00606 0.000564 11.67664 1.569094 530.3685 128.0059 4S-6/4S 0.942492 0.103514 124.9464 6.645647 7529.139 436.3087 8.094 4S-6/wt 0.96566 0.012454 1039.148 45.0631 930.154 30.78326 4S-9/4S 2.145552 0.116144 122.5609 12.77087 17624.7 1918.137 24.953 4S-9/wt 0.046796 0.005117 66.13857 3.761583 706.312 40.32513
(196) TABLE-US-00012 Avg Avg Avg Sdev Avg Sdev Subtrate/ k.sub.Hydro Sdev k.sub.cat Sdev K.sub.M K.sub.M Avg Sdev K.sub.H K.sub.H Target (Hz) k.sub.Hydro (Hz) k.sub.cat (uM) (uM) k.sub.cat/K.sub.M k.sub.cat/K.sub.M (uM) (uM) 4S-6/ 0.017818 0.00142 2.004458 0.088893 13114.69 1024.808 153.1223 5.537512 116.3542 10.43325 GGG 4S 2A-6/ 0.234725 0.075997 1.73442 0.07535 1111.343 272.4078 1610.214 308.4481 148.9406 62.62089 GGG 2A 4S-9/ 0.008016 0.001432 3.178356 0.390314 69450.39 12268.67 46.06877 3.27769 173.5925 32.49074 GGG 4S 2A-9/ 0.035062 0.002733 1.586937 0.153716 1484.283 193.4346 1072.459 45.49198 32.78664 4.076002 GGG 2A 4S-9/ 0.000173 5.65E06 0.057042 0.006454 97552.35 13438.31 0.587134 0.048202 294.8601 18.51321 GGG wt
DISCUSSION
(197) We applied a modified yeast display selection strategy to evolve highly active eSrtA into reprogrammed, orthogonal variants eSrtA(2A-9) and eSrtA(4S-9) with a 51,000- or 120-fold change in substrate specificity, respectively. eSrtA(2A-9) and eSrtA(4S-9) both have catalytic activity comparable to that of eSrtA, but strongly prefer LAXTG (SEQ ID NO: 9) and LPXSG (SEQ ID NO: 8) substrates, respectively, over the wild-type LPXTG (SEQ ID NO: 2) substrate. Substrate specificity profiling revealed that eSrtA(2A-9) strongly prefers the novel substrate LAXTG (SEQ ID NO: 9) to the next most active peptide mutant at position 2, LSETG (SEQ ID NO: 79), while eSrtA(4S-9) showed pronounced acceptance of LPXCG (SEQ ID NO: 84) and LPXAG (SEQ ID NO: 82) substrates in addition to LPXSG (SEQ ID NO: 8). Mutational dissection of eSrtA(2A-9) and eSrtA(4S-9) revealed the importance of residue 104 on enzyme activity and specificity at sorting motif position 2. Likewise, we identified that the combination of residues 104, 118 and 182 strongly determined the activity and specificity at position 4 in the sorting motif.
(198) We demonstrated the utility of SrtA reprogramming by showing that eSrtA(4S-9), unlike eSrtA or wild-type SrtA, is capable of modifying the human protein fetuin A in unmodified human plasma with high efficiency and specificity. In addition, we used these enzymes to synthesize the bioconjugates Alexa Fluor 750-LAETG-FGF1-PEG (SEQ ID NO: 5) and Alexa Fluor 750-LAETG-FGF2-PEG (SEQ ID NO: 5) using a set of four different PEG building blocks, and to simultaneously and orthogonally functionalize GGG-linked surfaces with target peptides.
(199) Consistent with the report of a promiscuous SrtA that processes XPETG (SEQ ID NO: 77) peptides by Piotukh and coworkers (47), we observed that eSrtA is capable of evolving significant changes in substrate specificity. Unlike this earlier report, however, here we demonstrate that SrtA may be further reprogrammed to possess fully altered, not merely broadened specificity. This capability is somewhat surprising given the mechanistic similarity between sortases and cysteine proteases (52) and the well-appreciated difficulty of the successful engineering of proteases with altered substrate specificities (68, 69). We hypothesize a number of possible explanations for this success. The combination of positive and negative selection strategy may be more effective than previous evolution methods. The relatively large library sizes used in this work (up to 10.sup.8 members), or our ability to precisely tune enzyme reaction conditions by adjusting the concentrations of exogenous substrates also likely played a role in successful sortase evolution. We also speculate that sortase itself is a privileged scaffold for the evolution of altered substrate specificity. Given the low degree of sequence homology among strain-specific sortase enzymes, the high degree of sorting motif homology observed across gram positive bacteria, and the observed difficulty in previous sortase substrate engineering studies, the most likely contributor to our successful sortase evolution efforts is a combination of these potential explanations.
(200) The high activity and specificity of eSrtA(4S-9) enabled the successful chemical modification in plasma of human fetuin A, the major carrier protein of calcium phosphate in vivo and a potent anti-inflammatory protein and inhibitor of soft tissue calcification (57). While fetuin A is traditionally difficult to purify away from its natively interacting partners (70), our strategy of site-specific reaction and pull-down afforded pure preparations of truncated fetuin A without detectable contaminants. Additionally, the modification of fetuin A raises a number of new research and therapeutic opportunities, including the in situ stabilization of the protein to potentially effect mortality outcomes in hemodialysis (58), the study of its proposed roles in hepatocyte invasion by P. berghei (59), and its role in insulin sensitivity (71).
(201) The milligram-scale synthesis of protein-PEG conjugates demonstrates the effectiveness of orthogonal transpeptidases in the synthesis of complex biomolecules. The combination of two orthogonal, high-activity enzymes enabled the facile synthesis of ten distinct fluorophore-FGF-PEG conjugates. Five of these were prepared at multi-milligram scale. Given the growing use of bioconjugates as human therapeutics (72) and the recent therapeutic interest in FGF1 as a treatment for diabetes (62), we anticipate that this technique may prove useful in the rapid generation and testing of a wide variety of protein-small molecule and protein-polymer constructs for use in research and therapeutic contexts.
(202) Finally, our use of orthogonal eSrtA variants for the synthesis of peptide-conjugated surfaces illustrates the potential utility of our evolved SrtA variants for novel materials syntheses. By enabling the specific and orthogonal conjugation of proteins and material surfaces, we anticipate that orthogonal evolved sortases will enable the construction of previously inaccessible materials containing multiple, homogenously immobilized proteins.
(203) The statistical rarity of a given peptide 5-mer within a typical proteome implies that a reasonably specific SrtA-derived transpeptidase is unlikely to react with more than a small number of targets. The applications achieved in this study, coupled with the generality of our eSrtA specificity changing strategy, suggest that it should be possible to reprogram sortases to selectively target other proteins of biological or therapeutic interest.
(204) Analysis of eSrtA Sequencing Results
(205) Table 6 shows the analysis of eSrtA round 3 sequencing results from experiments in example 5. Amino acid-level mutations relative to eSrtA are listed along with their multiplicities (first column). The canonical eSrtA(2A-3) and eSrtA(4S-3) variants are shown in bold.
(206) TABLE-US-00013 TABLE 6 Analysis of eSrtA round 3 sequencing results. 2A Round 3 Sequencing 2 K84R F122S D124G K134R K145E K162R V168I K177R 1 K84R F122S D124G K134R M155V K162R K177R 1 K84R F122S D124G K134R K138I K145E K162R K177G 1 K84R F122S D124G K134R M155I K162R K177G 1 K84R F122S D124G K134R M155V K162R K177G 4S Round 3 Sequencing 8 N98S A104T A118T F122S D124G 2 H98S A104T A118T F122S 1 N98S A104T E106G A118T F122S 1 N98S A104T A118T F122S 1 N98S A104T N107S A118T F122S D124G 1 I67V K69E N98S A104T A118T F122S 1 N98S A104T A118T F122S D124G 1 N98S A104T A118T F122S D124G 1 N98S A104T A118T F122S D124G 1 N98S A104T A118T F122S D124G 1 H98S A104T I115V A118T F122S D124G 2A Round 3 Sequencing 2 I182F 1 I182F 1 I182F 1 I182F 1 I182F 4S Round 3 Sequencing 8 K134R K173E K177E I182V 2 K134R K173E K177E 1 K134R K173E K177E K205T 1 K134R K173E K177E E190G 1 K134R K173E K177E I182V 1 K134R K173E K177E I182V 1 K134R K173E K177D 1 K134R F144L K173E K177E 1 K134R T150A K173E K177E 1 R125H K134R K173E K177D 1 K134R K173E K177D
(207) Table 7 shows the analysis of eSrtA round 4 sequencing results from experiments in example 5. Amino acid-level mutations relative to eSrtA are listed along with their multiplicities (first column). The canonical eSrtA(2A-4) and eSrtA(4S-4) variants are shown in bold.
(208) TABLE-US-00014 TABLE 7 Analysis of eSrtA round 4 sequencing results. 2A Round 4 Sequencing 12 A104H K162R V168I I182V 1 A104H E106D K162R V168I I182F 1 A104H I182V 1 A81V A104H I182V 1 S102C A104H K162R V168I I182V 1 I182F 1 A104H S157I K162R V168I 1 A104H K162R V168I 1 K62R A81S A104H I182V 4S Round 4 Sequencing 4 A118T F122S I182V 4 F122S I182V 3 A104V A118T F122S I182V 1 V101A A104T F122S I182V 1 A118T F122S G147D I182V 1 A104V A118T F122C 1 A104I A118T F122S K162R I182V 1 F122S I182L 1 A104V A118T F122C I182V 1 A104V F122S I182V 1 A61T A118T F122S I182V 1 F122S K162R I182V
(209) Table 8 shows the analysis of eSrtA round 5 sequencing results from experiments in example 5. Amino acid-level mutations relative to eSrtA are listed along with their multiplicities (first column). The canonical eSrtA(2A-5) and eSrtA(4S-5) variants are shown in bold.
(210) TABLE-US-00015 TABLE 8 Analysis of eSrtA round 5 sequencing results. 2A Round 5 Sequencing 34 R99H A104H K138I K162R I182V 10 A104H K138I K162R V168I I182V 1 A104B K162R I182V E189V 4S Round 5 Sequencing 13 N98D A104V A118S F122A I182V 12 N98D A104V A118S F122S I182V 4 A104V A118S F122S I182V 3 A104V A118S F122A I182V 2 A104V A118T F122H I182V 1 N98A A104V A118S F122S I182V 1 A104V A118S F122A I182V 1 N98D A104V A118T F122A K134R I182V 1 A104V A118S F122S I182V K206R 1 N98D A104V A118S F122A I182V K206* 1 K71R A104V A118S F122A I182V E189V 1 N98D A104V A118S F122S A135S I182V 1 A104V A118S F122S K175S I182V 1 S102C A104V A118T F122S I182V 1 N98A A104V A118S F122A K134R I182V 1 N98D A104V A118S F122H I182V 1 N98D A104V A118T F122A K134R I182V E189K 1 S102C A104V A118T F122A I182V
(211) Table 9 shows the analysis of eSrtA round 6 sequencing results from experiments in example 5. Amino acid-level mutations relative to eSrtA are listed along with their multiplicities (first column). The canonical eSrtA(2A-6) and eSrtA(4S-6) variants are shown in bold.
(212) TABLE-US-00016 TABLE 9 Analysis of eSrtA round 6 sequencing results. 2A Round 6 Sequencing 7 A104H K138V K162R I182V 5 R99K A104H K138V N160K K162R I182V 3 R99L A104H K138L K162R I182V 3 A104H K138I K145R N160K K162R I182V 3 A104H K138V N160R K162R I182V 2 A104H K138L N160K K162R I182V 2 R99K A104H K138L K162R I182V E189M 2 R99L A104H K138L K162A I182V 2 A104H K138I K162R I182V 1 A104H K138T N160K K162R I182V 1 A104H K138L K162R I182V 1 R99T A104H K138V K162H I182V 1 A104H K138M N160T K162R I182V 1 A104H K138L K162R Q172H I182V 1 R99H A104H K138M K162R I182V E189N 1 R99L A104H K138M K162R I182V 1 R99T A104H K138L K162R I182V 1 R99T A104H K138V K162R I182V 1 A104H K138V K162R I182V 1 R99K A104H K138M I182V 1 R99T A104H K138Y K162R I182V 1 R99K A104H K138L K162R I182V 1 A104H K138Y K162R I182V 1 A73V A104H K138V N160K K162R I182V 1 R99L A104H K138L K162R I182V E189Q 1 A104H K138V N160T K162R I182V 1 A104H K138P N160K K162R I182V 4S Round 6 Sequencing 7 N98D A104V A118S F122A K134G I182V E189V 5 N98D A104V A118S F122A K134R I182V 2 N98D A104V A118S F122A K134H D176N I182V E189T 2 N98D A104V A118T F122A R134H I182V E189T 2 N98D A104V A118T F122A K134R I182V E189S T203I 1 N98D A104V A118T F122A K134R I182V E189L 1 N98D A104V E106G A118T F122A K134R A165S I182V 1 N98D A104V A118T F122A K134R I182V E189F 1 N98D A104V A118T F122A K134R K145L I182V E189A 1 N98D A104V A118S F122A K134P I182V E189P 1 N98D A104V A118T F122A K134R I182V E189T 1 N98D A104V A118T F122A K134R I182V E189A 1 N98D A104V A118T F122A K134R A136V I182V E189P 1 N98D A104V A118T F122A K134R I182V E189S 1 N98D A104V A118T F122A K134R I182V E189V 1 N98D A104V A118T F122A K134R I182V E189R 1 N98D A104V A118T F122A K134R I182V E189I 1 N98D A104V A118T F122A K134R A165K I182V
(213) Table 10 shows the analysis of eSrtA round 9 sequencing results from experiments in example 5. Amino acid-level mutations relative to eSrtA are listed along with their multiplicities (first column). The canonical eSrtA(2A-9) and eSrtA(4S-9) variants are shown in bold. Stop codons are denoted by an asterisk.
(214) TABLE-US-00017 TABLE 10 Analysis of eSrtA round 9 sequencing results. 2A Round 9 Sequencing 4 S102C A104H E105D K138P K152I N160K 3 S102C A104H E105D K138P K152I N160K 2 S102C A104H E105D K138P K152I N160K 2 A104H E105D K138P K152I N160K 2 S102C A104H E105D K138P K152I N160K 2 A104H E105D K134R K138L K152I N160K 2 S102C A104H E105D K138P K152I N160K 1 A104H E105D K138L K152I N160K 1 S102C A104H E105D K138P K152I N160K 1 S102C A104H E105D K138P K152I N160K 1 A104H E105D K138P K152I N160K 1 S102C A104H E105D K137R K138P K152I N160K 1 A104H E105D F122Y K138L K152I N160K 1 Q64H A104H E105D K138P K152I N160K 1 S102C A104H E105D K138P K152I N160K 1 A104H N107D K138P K152I N160K 1 S102C A104H F122Y K138P K152I N160K 1 A104H E105D K137R K138P K152I N160K 1 A104H E105D K138L K152I N160K 1 S102C A104H E105D K138L K152I N160K 1 K71Q A104H E105D K138P K152I N160K 1 S102C A104H E105D F122Y K138L K152I N160K 1 A104H E105D N107D K138P K152I N160K 1 S102C A104H E105D F122Y K138L K152I N160K 1 S102C A104H E105D K138P K152I N160K 1 S102C A104H E105D K138P K152I N160K 1 A104H E105D K138L K152I N160K 1 S102C A104H E105D F122Y K138P K152I N160K 1 S102C A104H K138P K152I N160K 1 A104H E105D N107D K138L K152I N160K 4S Round 9 Sequencing 11 N98D S102C A104V A118T F122A 8 S70T E77V N98D S102C A104V A118T F122A 3 L97I N98D S102C A104V A118T F122A 2 N98D S102C A104V N107D A118T F122A D124G 2 N98D S102C A104V N107D A118T F122A 1 N98D S102C A104V A118T F122A 1 S70T E77V N98D S102C A104V A118T F122A 1 N98D S102C A104V A118T F122A 1 L97I N98D S102C A104V A118T F122A 1 Q64H S70T E77V N98D S102C A104V A118T F122A 1 N98D S102C A104V A118T F122A 1 X84I N98D S102C A104V A118T F122A 1 S70T E77V N98D S102C A104V A118T F122A 1 X84E N98D S102C A104V A118T F122A 1 E77V N98D S102C A104V A118T F122A 1 X84I N98D S102C A104V N107D A118T F122A 1 N98D S102C A104V A118T F122A 1 N98D S102C A104V A118T F122A D124Q 1 N98D S102C A104V A118T F122A 1 S70T E77V N98D S102C A104V A118T F122A 1 S70T E77V N98D S102C A104V A118T F122A 1 N98D S102C A104V N107D A118T F122A D124G 2A Round 9 Sequencing 4 K162H T164N K173E I182V T196S 3 K162H T164N I182V T196S 2 K162R I182V T196S 2 K162H T164N I182V T196S 2 K162R I182V T196A 2 K162R I182V 2 K162R T164S I182V 1 K162H T164N K173E I182V T196S 1 K162H T164N K173E I182V 1 K162R K173E I182V 1 K162R K173E I182V 1 K162R T164N I182V T196A 1 K162P I182V 1 K162H T164N I182V 1 K162R I182V N188F 1 K162H T164N I182V T196A 1 K162R T164S I182V 1 K162R I182V 1 K162H T164N I182V 1 K162R T164S I182V 1 K162R I182V T196S 1 K162R T164S I182V 1 K162H T164N I182V T196A 1 K162R K173E I182V 1 K162R T164S I182V T196A 1 K162R I182V 1 K162R I182V 1 K162R I182V T196S 1 K162H T164N I182V T196A 1 K162R I182V 4S Round 9 Sequencing 11 K134R F144L I182V E189F 8 N127Y K134R F144L I182V E189F K206* 3 K134R F144L I182V E189F 2 K134R F144L I182V E189F 2 K134R F144L N148I I182V E189F 1 N127Y K134R F144L N148I I182V E189F K206* 1 K134R F144L I182V E189F 1 K134R F144L N148I I182V E189S 1 K134R F144L I182V E189F F200Y K206* 1 N127Y K134R F144L I182V E189F 1 N127Y K134R F144L I182V E189F E190V 1 N127Y K134R F144L I182V E189F K206* 1 N127Y K134R I182V E189F K206* 1 K134R F144L I182V E189F 1 K134R F144L I182V E189F K206* 1 K134R F144L I182V E189F 1 K134R F144L D176G I182V E189F F200Y K206* 1 K134R F144L I182V E189F 1 N127Y K134R F144L I182V E189F 1 K134R F144L I182V E189I 1 N127Y K134R F144L K175I I182V E189F 1 K134R F144L I182V E189F K206*
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(216) All publications, patents and sequence database entries mentioned herein, including those items listed in the Summary, Brief Description of the Drawings, Detailed Description, and Examples sections, are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.
EQUIVALENTS AND SCOPE
(217) Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the above description, but rather is as set forth in the appended claims.
(218) In the claims articles such as a, an, and the may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include or between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention also includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.
(219) Furthermore, it is to be understood that the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the claims or from relevant portions of the description is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Furthermore, where the claims recite a composition, it is to be understood that methods of using the composition for any of the purposes disclosed herein are included, and methods of making the composition according to any of the methods of making disclosed herein or other methods known in the art are included, unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise.
(220) Where elements are presented as lists, e.g., in Markush group format, it is to be understood that each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It is also noted that the term comprising is intended to be open and permits the inclusion of additional elements or steps. It should be understood that, in general, where the invention, or aspects of the invention, is/are referred to as comprising particular elements, features, steps, etc., certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements, features, steps, etc. For purposes of simplicity those embodiments have not been specifically set forth in haec verba herein. Thus for each embodiment of the invention that comprises one or more elements, features, steps, etc., the invention also provides embodiments that consist or consist essentially of those elements, features, steps, etc.
(221) Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. It is also to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values expressed as ranges can assume any subrange within the given range, wherein the endpoints of the subrange are expressed to the same degree of accuracy as the tenth of the unit of the lower limit of the range.
(222) In addition, it is to be understood that any particular embodiment of the present invention may be explicitly excluded from any one or more of the claims. Where ranges are given, any value within the range may explicitly be excluded from any one or more of the claims. Any embodiment, element, feature, application, or aspect of the compositions and/or methods of the invention, can be excluded from any one or more claims. For purposes of brevity, all of the embodiments in which one or more elements, features, purposes, or aspects is excluded are not set forth explicitly herein.