FKBP DOMAIN WITH TRANSGLUTAMINASE RECOGNITION SITE
20190381181 ยท 2019-12-19
Assignee
Inventors
Cpc classification
A61K47/65
HUMAN NECESSITIES
C07K2319/35
CHEMISTRY; METALLURGY
C12N2740/14022
CHEMISTRY; METALLURGY
International classification
Abstract
The present disclosure relates to a recombinant transglutaminase (TG) substrate having an amino acid sequence of the FKBP domain of an FKBP polypeptide, wherein the insert-in-flap (IF) domain thereof is, at least in part, replaced by an amino acid sequence (Q-tag) of 5 to 20 amino acids with a sequence having at least 80% sequence identity to the YRYRQ portion of the peptide sequence X.sub.1-YRYRQ-X.sub.2 (SEQ ID NO. 1), and wherein said TG substrate is a substrate for the TG function of the Kutzneria albida TG. The present disclosure furthermore relates to uses of said substrate.
Claims
1. A recombinant transglutaminase (TG) substrate according to the following general formula I
(F*-L).sub.y-X(I) wherein F* is selected from an amino acid sequence of the FKBP domain of an FKBP polypeptide, wherein the insert-in-flap (IF) domain thereof is, at least in part, replaced by an amino acid sequence (Q-tag) of 5 to 20 amino acids, the Q-tag comprising a sub-sequence of 5 contiguous amino acids having at least 80% sequence identity to the YRYRQ portion of the peptide sequence X.sub.1-YRYRQ-X.sub.2 (SEQ ID NO. 1), wherein X.sub.1 and X.sub.2 are absent or constitute linker amino acids; L is absent or is selected from a linker amino acid sequence; and X is a protein of interest; y is an integer of between 1 and 100, and wherein said TG substrate is a substrate for the TG function of the Kutzneria albida TG according to SEQ ID No. 23.
2. The recombinant transglutaminase (TG) substrate according to claim 1, wherein said FKBP domain is selected from a eukaryotic or bacterial FKBP polypeptide selected from FKBP12, AIP, AIPL1, FKBP1A, FKBP1B, FKBP2, FKBP3, FKBP5, FKBP6, FKBP7, FKBP8, FKBP9, FKBP9L, FKBP10, FKBP11, FKBP14, FKBP15, FKBP52, LOC541473, and SLYD, and homologs of the FKBP domains thereof.
3. The recombinant transglutaminase (TG) substrate according to claim 1, wherein said FKBP domain comprises between about 120 to 170 of the N-terminal amino acids of said FKBP polypeptide.
4. The recombinant transglutaminase (TG) substrate according to claim 1, wherein said FKBP domain comprises the N-terminal amino acids 1 to 64 and 123 to 149 of the SLYD polypeptide, and wherein amino acids 65 to 122 are replaced by said Q-tag.
5. The recombinant transglutaminase (TG) substrate according to claim 1, wherein said linker sequence L comprises between 1 to 20 amino acids, wherein said amino acids do not interfere essentially with the FKBP domain and/or the protein of interest.
6. The recombinant transglutaminase (TG) substrate according to claim 1, wherein said protein of interest is selected from an enzyme, an antigen, such as a viral protein, an antibody or fragment thereof, and other immunological binding partners.
7. An in vitro method for labelling a protein of interest, comprising a) providing the recombinant transglutaminase (TG) substrate according to claim 1 being attached to a protein of interest, b) providing an effective amount of the transglutaminase of Kutzneria albida, according to SEQ ID No. 23, c) providing a suitable label linked comprising an alkyl-amine group, and d) contacting said components according to a) to c), whereby said transglutaminase attaches said label to said substrate.
8. The method according to claim 7, wherein said transglutaminase of Kutzneria albida is recombinantly produced.
9. The method according to claim 7, wherein said label is selected from an enzyme, biotin, a radioactive group, a dye, an isotope, a chemiluminescent label, and a metal.
10. The method according to claim 7, wherein said labeling is achieved in a stoichiometric ratio of label and protein of interest at about 1:1.
11. The method according to claim 7, wherein said protein of interest is selected from an enzyme, an antigen, a viral protein, an antibody or fragment thereof, and other immunological binding partners.
12. A pharmaceutical or diagnostic composition comprising at least one labeled protein of interest as produced according to a method according to claim 7, together with pharmaceutically acceptable carrier compounds.
13. The pharmaceutical or diagnostic composition according to claim 12, wherein said protein of interest is labelled at a stoichiometric ratio of label and protein of interest of about 1:1.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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BRIEF DESCRIPTION OF THE SEQUENCE IDENTIFIERS
[0034] SEQ ID NO: 1 to 22 show the sequences of the Q-tag motifs as identified in the context of the present invention, wherein the N-terminal X is as X.sub.1 above, and the C-terminal X is as X.sub.2 as above.
[0035] SEQ ID NO: 23 shows the amino acid sequence of the transglutaminase from Kutzneria albida (database no. WP_030111976).
[0036] SEQ ID NO: 24 shows the amino acid sequence of slyD of E. coli.
[0037] SEQ ID NO: 25 shows the amino acid sequence of slyD of Cyclobacteriaceae bacterium AK24.
[0038] SEQ ID NO: 26 shows the amino acid sequence of slyD of Bacteroidales bacterium CF.
[0039] SEQ ID NO: 27 shows the amino acid sequence of the FKBP domain of slyD of E. coli.
[0040] SEQ ID NO: 28 shows the amino acid sequence of the FKBP domain of FKBP16 of E. coli.
[0041] SEQ ID NO: 29 shows the amino acid sequence of the FKBP domain of slyD of Thermus thermophilus.
[0042] SEQ ID NO: 30 to 51 show the sequences of the Q-tag motifs as identified in the context of the present invention, wherein the N-terminal and C-terminal amino acid is one exemplary glycine linker.
[0043] SEQ ID NO: 52 shows the amino acid sequence of the KalbTG glutamine donor sequence that was recombinantly grafted onto the FKBP domain of SlyD.
[0044] SEQ ID NO: 53 shows the amino acid sequence of a transglutaminase lysine donor sequence (K-tag).
[0045] SEQ ID NO: 54 shows the SlyD amino acid sequence.
[0046] SEQ ID NO: 55 shows the amino acid sequence of SlyD with a MTG Q-tag.
[0047] SEQ ID NO: 56 shows the amino acid sequence of SlyD with a KalbTG Q-tag.
[0048] SEQ ID NO: 57 shows the SlpA amino acid sequence.
[0049] SEQ ID NO: 58 shows the main HTLV antigen and viral envelope glycoprotein amino acid sequence gp21.
[0050] SEQ ID NO: 59 shows the amino acid sequence of modified the gp21 ectodomain polypeptide sequence, engineered for better solubility, stability and reactivity in the immunoassay.
[0051] SEQ ID NO: 60 shows the amino acid sequence of the recombinant fusion protein TtSlyQD-Xagp21-8H.
[0052] SEQ ID NO: 61 shows the amino acid sequence of the recombinant fusion protein TtSlyD-Xagp21-8H.
[0053] SEQ ID NO: 62 shows the amino acid sequence of the recombinant fusion protein TtSlyKQDSlpA-Xa-gp21-8H.
DETAILED DESCRIPTION OF THE INVENTION
[0054] In view of the above, it is an object of the present invention to provide new tools and methods in order to provide for an efficient and controlled labelling of antigens and/or antibodies or other immunological binding partners (proteins of interest) in the context of immunological diagnostic and/or therapeutic systems and methods. Other aspects and objects will become apparent for the person of skill upon reading the following more detailed description of the invention.
[0055] According to a first aspect thereof, the above objects are solved by the provision of a recombinant transglutaminase (TG) substrate according to the following general formula I
(F*-L).sub.y-X(I).
[0056] In said formula (I), F* is selected from an amino acid sequence of the FKBP domain of an FKBP polypeptide, preferably comprising an insert-in-flap (IF) domain that in the unmodified polypeptide is inserted internally as a guest into a surface loop of the host domain, which is the prolyl isomerase of the FK506 binding protein (FKBP) type. Nevertheless, the invention also includes FKBP domains that naturally (unmodified) do not include an insert-in-flap (IF) domain, wherein the Q-tag is inserted at the homologous position in the enzyme.
[0057] Said insert-in-flap (IF) domain is, at least in part, replaced by an amino acid sequence (Q-tag) having a length of 5 to 20 amino acids comprising a sub-sequence of 5 contiguous amino acids, the sub-sequence having at least 80% sequence identity to the peptide sequence X.sub.1-YRYRQ-X.sub.2 (SEQ ID NO. 1), specifically to the YRYRQ portion of the peptide sequence X.sub.1-YRYRQ-X.sub.2 (SEQ ID NO. 1), wherein X.sub.1 and X.sub.2 are absent or constitute linker amino acids. In a specific embodiment, the IF domain is, at least in part, replaced by X.sub.1-YRYRQ-X.sub.2 (SEQ ID NO. 1) (Q-tag) having a length of 5 to 15 or 5 to 20 amino acids, the Q-tag comprising a contiguous sub-sequence of five amino acids (i.e. a sub-sequence consisting of five contiguous amino acid residues), the sub-sequence having at least 80% sequence identity to the YRYRQ portion of (in) the peptide sequence X.sub.1-YRYRQ-X.sub.2(SEQ ID NO. 1), wherein X.sub.1 and X.sub.2 are absent or constitute linker amino acids.
[0058] In a more specific embodiment, only X.sub.1 is absent and the length of the peptide sequence X.sub.1-YRYRQ-X.sub.2 is 6 to 15 or 6 to 20 amino acids. In an even more specific embodiment, X.sub.2 comprises (or may consist of) an arginine residue directly following the YRYRQ sub-sequence or a subsequence having at least 80% sequence identity to the YRYRQ portion.
[0059] In yet a further very specific embodiment, in the sub-sequence consisting of five contiguous amino acid residues having at least 80% sequence identity to the YRYRQ portion of the peptide sequence X.sub.1-YRYRQ-X.sub.2 (SEQ ID NO. 1), a glutamine residue is on a selected position of the sub-sequence, the position in the sub-sequence being selected from the group consisting of the third position, the fourth position, the fifth position, and a combination thereof.
[0060] In yet a further very specific embodiment, in the sub-sequence consisting of five contiguous amino acid residues having at least 80% sequence identity to the YRYRQ portion of the peptide sequence X.sub.1-YRYRQ-X.sub.2 (SEQ ID NO. 1), an arginine residue is on a selected position of the sub-sequence, the position in the sub-sequence being selected from the group consisting of the fourth position, the fifth position, and a combination thereof.
[0061] It is further understood that, according to the invention, the linker amino acids X.sub.1 and X.sub.2, if present, are selected independently from each other.
[0062] Preferred are Q-tags according to the present invention that re selected from the sequences X.sub.1-YRYRQ-X.sub.2 (SEQ ID NO: 1), X.sub.1-RYRQR-X.sub.2 (SEQ ID NO: 2), X.sub.1-RYSQR-X.sub.2 (SEQ ID NO: 3), X.sub.1-FRQRQ-X.sub.2 (SEQ ID NO: 4), X.sub.1-RQRQR-X.sub.2 (SEQ ID NO: 5), X.sub.1-FRQRG-X.sub.2 (SEQ ID NO: 6), X.sub.1-QRQRQ-X.sub.2 (SEQ ID NO: 7), X.sub.1-YKYRQ-X.sub.2 (SEQ ID NO: 8), X.sub.1-QYRQR-X.sub.2 (SEQ ID NO: 9), X.sub.1-YRQTR-X.sub.2 (SEQ ID NO: 10), X.sub.1-LRYRQ-X.sub.2 (SEQ ID NO: 11), X.sub.1-YRQSR-X.sub.2 (SEQ ID NO: 12), X.sub.1-YQRQR-X.sub.2 (SEQ ID NO: 13), X.sub.1-RYTQR-X.sub.2 (SEQ ID NO: 14), X.sub.1-RFSQR-X.sub.2 (SEQ ID NO: 15), X.sub.1-QRQTR-X.sub.2 (SEQ ID NO: 16), X.sub.1-WQRQR-X.sub.2 (SEQ ID NO: 17), X.sub.1-PRYRQ-X.sub.2 (SEQ ID NO: 18), X.sub.1-AYRQR-X.sub.2 (SEQ ID NO: 19), X.sub.1-VRYRQ-X.sub.2 (SEQ ID NO: 20), X.sub.1-VRQRQ-X.sub.2 (SEQ ID NO: 21), and X.sub.1-YRQRA-X.sub.2 (SEQ ID NO: 22), wherein X.sub.1 and X.sub.2 are as above.
[0063] It was surprisingly found that the two parts of the FKBP domain upstream and downstream insertion of the IF domain function as structurally rather rigid and precise scaffold or collar for the sequence that is inserted into and/or replaces the IF domain part (head, Q-tag). Because of this, the presentation and orientation of the insertion/replacement reliably does not substantially interfere with any other function of the other components of the substrate, in particular the function(s) of the protein of interest (X). Furthermore, the presentation of thein this caseTG substrate binding site (Q-tag) leads to a highly controlled stoichiometric binding of the label, and hence labelling of the protein of interest.
[0064] In formula (I), L is absent or is selected from a linker amino acid sequence. Preferably, said linker sequence L comprises between 1 to 20 amino acids and more preferred said amino acids do not interfere essentially with the function(s) of the FKBP domain (in particular the replacement/insertion) and/or the protein (s9 of interest (e.g. immunological functions).
[0065] X designates the protein of interest; that is preferably selected from an enzyme, an antigen, such as a viral protein, an antibody or fragment thereof, and other immunological binding partners.
[0066] In formula (I), y is an integer of between 1 and 100, thus, several marker groups F*-L can be attached to the protein of interest.
[0067] Preferably, the recombinant transglutaminase (TG) substrate according to the invention can furthermore comprise protein tags for purification and/or immobilization, for example at the N_ and/or C-terminus, like biotin, maltose or his-tags(s).
[0068] Preferably, the different components of the substrate F*, L and/or X are covalently attached with each other in order to allow the controlled labeling of the substrate using the TG activity. The substrate can be recombinantly produced as a fusion protein, and expressed and purified from hosts cells, such as, for example, bacterial or yeast host cells. Respective methods are well known to the person of skill. The components can also be produced (e.g. synthesized) separately or in parts, and are then subsequently joined, either covalently or conjugated, depending on the circumstances and the desired purpose(s) thereof.
[0069] According to the invention, the inventive TG substrate is a substrate for the transglutaminase (TG) function of the Kutzneria albida TG according to SEQ ID No. 23, or a homologous protein that is identical to at least 80%, preferably to at least 90%, more preferably to at least 95, 98 or 99% to the amino acid sequence thereof, and exhibit substantial TG activity as described herein. Preferably, the TG polypeptide or a part thereof having a substantial transglutaminase function or activity is cloned and recombinantly produced in hosts cells, and subsequently (at least in part) purified, depending on the circumstances and the desired purpose(s) thereof. Respective methods are well known to the person of skill. The TG activity can be measured using assays that are also well known to the person of skill. Recombinant TG substrate in the context of the invention shall mean that the substrate as a whole does not occur in nature.
[0070] The FKBP domain as used for the recombinant transglutaminase (TG) substrate according to the present invention is preferably selected from a eukaryotic or bacterial FKBP polypeptide selected from FKBP12, AIP, AIPL1, FKBP1A, FKBP1B, FKBP2, FKBP3, FKBP5, FKBP6, FKBP7, FKBP8, FKBP9, FKBP9L, FKBP10, FKBP11, FKBP14, FKBP15, FKBP52, LOC541473, and SLYD, and homologs of the FKBP domains thereof that are identical to at least 80%, preferably to at least 90%, more preferably to at least 95, 98 or 99% to the amino acid sequence thereof, and are suitable for the insertion of a Q-tag. Respective domains can be analyzed for their suitability as described herein, and in the literature, e.g. using alignment programs, in particular to identify structural alignments.
[0071] In an embodiment, a FKBP domain as used for the recombinant transglutaminase (TG) substrate according to the present invention comprises an amino acid sequence of a polypeptide with PPIase activity. The polypeptide with PPIase activity is of prokaryotic or eukaryotic origin, or the polypeptide with PPIase activity as an artificial variant (mutant) thereof. An artificial variant of a polypeptide with PPIase activity can be generated by a technique according to the state of the art of protein engineering. According to the invention, an artificial variant of a polypeptide with PPIase activity is characterized e.g. by replacement, addition or deletion of one or more amino acids. In a specific embodiment, PPIase activity and/or chaperone function of the FKBP domain as used for the recombinant transglutaminase substrate according to the present invention is/are preserved in an artificial variant.
[0072] The catalytic activity of human FKBP12 as a prolyl isomerase is high towards short peptides, but very low in proline-limited protein folding reactions. In contrast, the SlyD proteins, which are members of the FKBP family, are highly active as folding enzymes. They contain an extra insertin-flap or IF domain near the prolyl isomerase active site. The excision of this domain did not affect the prolyl isomerase activity of SlyD from Escherichia coli towards short peptide substrates but abolished its catalytic activity in proline-limited protein folding reactions. The reciprocal insertion of the IF domain of SlyD into human FKBP12 increased its folding activity 200-fold and generated a folding catalyst that is more active than SlyD itself. The IF domain binds to refolding protein chains and thus functions as a chaperone module.
[0073] In E. coli, it is believed that amino acids 1 to 69 of SLYD form the first part of the PPIase domain, amino acids 76 to 120 form the IF-chaperone (domain), and amino acids 129 to 151 form the second part of the PPIase domain. Thus, amino acids 1 to 151 form the FKBP-domain. Amino acids 152 to 196 are involved in metal binding.
[0074] Preferred is the recombinant transglutaminase (TG) substrate according to the present invention, wherein said FKBP domain has a length of between about 120 to 170, preferably between about 130 to 160, and most preferred between about 145 to 155 of the N-terminal amino acids of said FKBP polypeptide. This includes the IF-domain sequence.
[0075] About in the context of the present invention shall mean+/10% of a given value.
[0076] Preferred is the recombinant transglutaminase (TG) substrate according to any one of claims 1 to 3, wherein said FKBP domain comprises the N-terminal amino acids 1 to 64 and 123 to 149 of the SLYD polypeptide, and wherein amino acids 65 to 122 are replaced by said Q-tag.
[0077] As mentioned above, the linker sequence L can comprise between 1 to 20 amino acids, preferably 1 to 10 amino acids, more preferred 1 to 5 amino acids wherein preferably said amino acids to not interfere essentially with the FKBP domain and/or the protein of interest. Preferred linker amino acids are small amino acids, like glycine or alanine. Amino acid linkers and their compositions are known in the art (see, e.g. Chichili et al. Linkers in the structural biology of proteinprotein interactions Protein Sci. 2013 February; 22(2): 153-167).
[0078] Proteins of interest in the context of the present invention are generally all proteins that can be labelled using the present technology. Preferred is the recombinant transglutaminase (TG) substrate according to the present invention, wherein said protein of interest is selected from an enzyme, an antigen, such as a viral protein, an antibody or fragment thereof, and other immunological binding partners. The present invention is of particular use for immunological reactions and assays, where quantification is desired.
[0079] Another aspect of the present invention then relates to an in vitro method for labelling a protein of interest, comprising a) providing the recombinant transglutaminase (TG) substrate according to the present invention comprising a protein of interest; b) providing an effective amount of the transglutaminase of Kutzneria albida (e.g. according to SEQ ID NO: 23) or a homolog thereof as described herein, c) providing a suitable label comprising an alkyl-amine group (amine donor), such as, for example, a lysine, and d) contacting said components according to a) to c), whereby said transglutaminase (activity) attaches said label to said substrate.
[0080] Preferred is a method according to the present invention, wherein said transglutaminase of Kutzneria albida or a homolog thereof as described herein is recombinantly produced, as described herein.
[0081] In this method according to the present invention, the substrate comprising the F* group comprising the Q-tag (optionally replacing the IF-domain region) is labelled using the activity of the transglutaminase of Kutzneria albida. Preferably, the label to be attached is selected from an enzyme, biotin, a radioactive group, a dye, such as a fluorescent dye, an isotope, and a metal. Said label is part of the label component/compound that comprises alkyl-amine group, such as, for example, a lysine. Most preferred, said label component/compound comprises an amine-donor tag (K-tag) having at least 80% sequence identity to the peptide sequence RYESK. Examples for preferred K-tags and their composition are described below and can also be found in the literature.
[0082] Preferred is a method according to the present invention, wherein said labeling is controlled and, for example, achieved in a stoichiometric ratio of label and protein of interest, for example at about 1:1. Multiple labeling can also be achieved using additional enzymes (e.g. other TGs than KalbTG) and respective other TG-substrates, in order to attach two or even multiple labels to a protein/proteins of interest.
[0083] Preferred is the method according to the present invention, wherein said protein of interest is selected from an enzyme, an antigen, such as a viral protein, an antibody or fragment thereof, and other immunological binding partners. The present invention is of particular use for immunological reactions and assays, where quantification is desired.
[0084] The choice of the protein of interest can also depend on the intended use of the piRNA molecules as used, either in therapy, research and/or diagnosis. Preferred is the method according to the invention, wherein said protein of interest is selected from protein involved/causing in cancer, neurological diseases, immunological diseases, allergy, metabolic diseases, fertility (e.g. reproductive rate or number), animal production (e.g. amount of meat or milk), protein essential for cell growth and/or development, for mRNA degradation, for translational repression, and/or for transcriptional gene silencing.
[0085] Another aspect of the present invention then relates to a pharmaceutical or diagnostic composition comprising at least one recombinant transglutaminase (TG) substrate according to the invention, together with pharmaceutically acceptable carriers and components, such as buffers. Pharmaceutical or diagnostic compositions for multiple labeling can also be obtained that comprise additional enzymes (e.g. other TGs than Kalb-TG) and respective other TG-substrates, in order to be able to attach two or even multiple labels to a protein/proteins of interest.
[0086] Another aspect of the present invention then relates to a pharmaceutical or diagnostic composition comprising at least one labeled protein of interest as produced according to a method according to the present invention, together with pharmaceutically acceptable carriers and components, such as buffers.
[0087] Preferred is the pharmaceutical or diagnostic composition according to the present invention, wherein said protein of interest is labelled at a stoichiometric ratio of label and protein of interest of, for example, about 1:1. Two or even several labels can also be attached to a protein/proteins of interest.
[0088] Another aspect of the present invention then relates to a diagnostic kit, comprising the diagnostic composition according to the present invention, optionally together with other components for performing an immunoassay. The kit can comprise, in joint or separate containers, a microbial recombinant transglutaminase, e.g. a purified microbial transglutaminase having at least 80% sequence identity to the Kutzneria albida microbial transglutaminase as described herein. The kit may further include substrates, such as a substrate including an amine-donor tag having at least 80% sequence identity to the peptide sequence RYESK. The kit can also include instructions for performing reactions (e.g. immunoassays) that require use of the substrate according to the invention, either labeled or un-labeled.
[0089] Another aspect of the present invention the relates to the use of the recombinant transglutaminase (TG) substrate, the pharmaceutical or diagnostic composition or the kit according to the present invention for labeling or in the labeling of a protein of interest, in particular in immunological reactions and assays, where a quantification is desired.
[0090] The methods of the present invention can be performed in vivo or in vitro, in particular in laboratory animals, or in culture.
[0091] The present invention will now be illustrated further in the following non-limiting examples, with reference to the accompanying figures. For the purposes of the invention, all publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.
EXAMPLES
Identification of the Position of the IF Domain
[0092] In principle, alignments of the primary structure of the amino acid sequences of FKBP domains can be used in order to identify the position of the IF domain to be replaced (or added), e.g. using the program Clustal Omega. An alternative, in particular with respect to non-bacterial FKBPs is the alignment of 3D structures (e.g. using the program PyMol), since the domain constitutes a structurally conserved element.
[0093] Specific examples are as follows:
[0094] SLYD_ECOLI FKBP-type peptidyl-prolyl cis-trans isomerase SlyD of Escherichia coli (strain K12). FKBP domain, IF domain double underlined.
TABLE-US-00001 (SEQIDNO:27) MKVAKDLVVSLAYQVRTEDGLVLVDESPVSAPLDYLHGHGSLISGLETA LEGHEVGDKFDVAVGANDAYGQYDENLVQRVPKDVFMGVDELQVGMRFL AETDQGPVPVEITAVEDDHVVVDGNHMLAGQNLKFNVEVVAIREATEEE LAHGHVHGAHDHHHDHDHD.
[0095] ECOLI FKBP-type 16 kDa peptidyl-prolyl cis-trans isomerase of Escherichia coli (strain K12). FKBP domain, IF domain double underlined.
TABLE-US-00002 (SEQIDNO:28) MSESVQSNSAVLVHFTLKLDDGTTAESTRNNGKPALFRLGDASLSEGLEQ HLLGLKVGDKTTFSLEPDAAFGVPSPDLIQYFSRREFMDAGEPEIGAIML FTAMDGSEMPGVIREINGDSITVDFNHPLAGQTVHFDIEVLEIDPALEA.
[0096] Peptidyl-prolyl cis-trans isomerase of Thermus thermophilus (strain HB8/ATCC 27634) FKBP domain, IF domain double underlined.
TABLE-US-00003 (SEQIDNO:29) MKVGQDKVVTIRYTLQVEGEVLDQGELSYLHGHRNLIPGLEEALEGREEG EAFQAHVPAEKAYGPHDPEGVQVVPLSAFPEDAEVVPGAQFYAQDMEGNP MPLTVVAVEGEEVTVDFNHPLAGKDLDFQVEVVKVREATPEELLHGHAH.
[0097] Analysis of the Q-tag sequences for KalbTG
[0098] Analysis of the KalbTG peptide substrates that were identified using labeling assays with a set of peptides comprising 5-mer amino acid sequences with three 3 N- and C-terminal glycine residues (G.sub.1 and G.sub.2) attached revealed a set of characteristics shared by these substrates. Specific examples were as follows:
TABLE-US-00004 (SEQIDNO:30) G.sub.1-YRYRQ-G.sub.2, (SEQIDNO:31) G.sub.1-RYRQR-G.sub.2, (SEQIDNO:32) G.sub.1-RYSQR-G.sub.2, (SEQIDNO:33) G.sub.1-FRQRQ-G.sub.2, (SEQIDNO:34) G.sub.1-RQRQR-G.sub.2, (SEQIDNO:35) G.sub.1-FRQRG-G.sub.2, (SEQIDNO:36) G.sub.1-QRQRQ-G.sub.2, (SEQIDNO:37) G.sub.1-YKYRQ-G.sub.2, (SEQIDNO:38) G.sub.1-QYRQR-G.sub.2, (SEQIDNO:39) G.sub.1-YRQTR-G.sub.2, (SEQIDNO:40) G.sub.1-LRYRQ-G.sub.2, (SEQIDNO:41) G.sub.1-YRQSR-G.sub.2, (SEQIDNO:42) G.sub.1-YQRQR-G.sub.2, (SEQIDNO:43) G.sub.1-RYTQR-G.sub.2, (SEQIDNO:44) G.sub.1-RFSQR-G.sub.2, (SEQIDNO:45) G.sub.1-QRQTR-G.sub.2, (SEQIDNO:46) G.sub.1-WQRQR-G.sub.2, (SEQIDNO:47) G.sub.1-PRYRQ-G.sub.2, (SEQIDNO:48) G.sub.1-AYRQR-G.sub.2, (SEQIDNO:49) G.sub.1-VRYRQ-G.sub.2, (SEQIDNO:50) G.sub.1-VRQRQ-G.sub.2, and (SEQIDNO:51) G.sub.1-YRQRA-G.sub.2.
[0099] For the KalbTG, the data revealed that an acyl-donor substrate including a 5-mer amino acid sequence having the formula Xaa.sub.1-Xaa.sub.2-Xaa.sub.3-Xaa.sub.4-Xaa.sub.5, where Xaa is any amino acid, generally complied with several design rules. First, each 5-mer sequence included at least one glutamine (Q). More particularly, at least one of the third, fourth, and fifth positions of the 5-mer sequence (i.e., Xaa.sub.3, Xaa.sub.4, and Xaa.sub.5) was a glutamine. Several sequences were observed that included a glutamine at each of the third and fifth positions. Furthermore, the observation was made that each 5-mer sequence included at least one arginine (R). More particularly, at least one of the fourth and fifth positions of the 5-mer sequence (i.e., Xaa.sub.4 and Xaa.sub.5) was an arginine.
Labeling Assays
1. Fluorescent Dye
[0100] For labeling assays, the chaperone SlyD from Thermus thermophilus (Universal Protein Resource (UniProt) Number Q5SLE7) was used as a labeling scaffold for KalbTG. The SlyD sequence is:
TABLE-US-00005 (SEQIDNO:29) MKVGQDKVVTIRYTLQVEGEVLDQGELSYLHGHRNLIPGLEEALEGREEG EAFQAHVPAEKAYGPHDPEGVQVVPLSAFPEDAEVVPGAQFYAQDMEGNP MPLTVVAVEGEEVTVDFNHPLAGKDLDFQVEVVKVREATPEELLHGHAH.
[0101] A KalbTG glutamine donor sequence (Q-tag, underlined) was recombinantly grafted onto the FKBP domain of SlyD, yielding the following polypeptide sequence:
TABLE-US-00006 (SEQIDNO:52) MKVGQDKVVTIRYTLQVEGEVLDQGELSYLHGHRNLIPGLEEALEGREEG EAFQAHVPAEKAYGAGSGGGGRYRQRGGGGGSSGKDLDFQVEVVKVREAT PEELLHGHAHHHHHHHH.
[0102] The 8X-histidine-tagged protein was produced in E. coli B121 Tuner and purified by standard Ni Sepharose-based immobilized metal ion affinity and size exclusion chromatography (HisTrap, Superdex 200; GE Healthcare). All peptides were synthesized via standard Fluorenylmethyloxycarbonyl (FMOC)-based solid phase peptide synthesis.
[0103] Labeled peptides were chemically synthesized to have (in order from N-terminus to C-terminus) a Z-protecting group (i.e., a carboxybenzyl group), a transglutaminase lysine donor sequence (K-tag), 8-amino-3,6-dioxaoctanoic acid (020c), peptide, and a Cy3 or Cy5 fluorescent dye. The primary chemical structure of the labeled peptides was:
TABLE-US-00007 (SEQIDNO:53) Z-RYESKG-O2Oc-EUEUEUEUEUEUEUEUEUEUEUEUEUEUEUEUEUEU EUEU-C(sCy3-MH)-OH.
[0104] Labeling reactions were performed for 15 minutes at 37 C. in the presence of 72 M substrate protein, 720 M label peptide and 1 M transglutaminase in 200 mM MOPS pH 7.2 and 1 mM EDTA. After incubation for 30 minutes at 37 C., 1 mM K-tag-Cy5 or -Cy3 was added and incubated for an additional 15 minutes at 37 C. The reaction was stopped by the addition of 50 mM TCA. Samples were taken between incubation steps and analyzed by SDS-PAGE, in-gel fluorescence (BioRad ChemiDoc gel documentation system, Cy3 and Cy5 LED and filter sets). Results are shown in
2. Ruthenium Label
[0105] A similar Q-tag construct was tested, consisting of a slyD Q-tag gp21 fusion (see
Production of Biotin- and Ruthenium Labeled TtSlyD-Gp21 (HTLV) Antigens
[0106] For further labeling assays, a recombinant fusion between the chaperone SlyD from Thermus thermophilus (Universal Protein Resource (UniProt) Number Q5SLE7), the HTLV viral envelope glycoprotein gp21 (Genbank Accession Number DQ224032.1) and, in one example, the chaperone SlpA from Escherichia coli (UniProt Number P0AEM0) was used as a labeling scaffold for MTG or KalbTG. The SlyD sequence is:
TABLE-US-00008 (SEQIDNO:54) MKVGQDKVVTIRYTLQVEGEVLDQGELSYLHGHRNLIPGLEEALEGREEG EAFQAHVPAEKAYGPHDPEGVQVVPLSAFPEDAEVVPGAQFYAQDMEGNP MPLTVVAVEGEEVTVDFNHPLAGKDLDFQVEVVKVREATPEELLHGHAH
[0107] A MTG or KalbTG glutamine donor sequence (Q-tag) was recombinantly grafted onto the FKBP domain of SlyD, yielding the following polypeptide sequences:
[0108] SlyD with MTG Q-tag:
TABLE-US-00009 (SEQIDNO:55) MKVGQDKVVTIRYTLQVEGEVLDQGELSYLHGHRNLIPGLEEALEGREEG EAFQAHVPAEKAYGAGSGGGGDYALQGGGGGSSGKDLDFQVEVVKVREAT PEELLHGHAH
[0109] SlyD with KalbTG Q-tag:
TABLE-US-00010 (SEQIDNO:56) MKVGQDKVVTIRYTLQVEGEVLDQGELSYLHGHRNLIPGLEEALEGREEG EAFQAHVPAEKAYGAGSGGGGYRYRQGGGGGSSGKDLDFQVEVVKVREAT PEELLHGHAH
[0110] The SlpA sequence is:
TABLE-US-00011 (SEQIDNO:57) MSESVQSNSAVLVHFTLKLDDGTTAESTRNNGKPALFRLGDASLSEGLEQ HLLGLKVGDKTTFSLEPDAAFGVPSPDLIQYFSRREFMDAGEPEIGAIML FTAMDGSEMPGVIREINGDSITVDFNHPLAGQTVHFDIEVLEIDPALEA
[0111] The main HTLV antigen and viral envelope glycoprotein sequence gp21 is:
TABLE-US-00012 (SEQIDNO:58) IVSSACNNSLILPPFSLSPVPTVGSRSRRAVPVAVWFVSALAMGAGVAGG ITGSMSLASGKSLLHEVDKDISQLTQAIVKNHKNLLKIAQYAAQNRRGLD LLFWEQGGLCKALQEQCCFLNITNSHVSILQERPPLENRVLTGWGLNWDL GLSQWAREALQTGITLVALLLLVILAGPCIRCPCRTMHP
[0112] The gp21 ectodomain polypeptide sequence, engineered for better solubility, stability and reactivity in the immunoassay, is:
TABLE-US-00013 (SEQIDNO:59) MSLASGKSLLHEVDKDISQLTQAIVKNHKNLLKIAQYAAQNRRGLDLLFW EQGGLAKALQEQAAFLNITNSHVSILQERPPLENRVLTGWGLNWDLGLSQ WAREALQTG
[0113] The recombinant fusion sequences used for the labeling assays were:
TABLE-US-00014 TtSlyQD-Xa-gp21-8H: (SEQIDNO:60) MKVGQDKVVTIRYTLQVEGEVLDQGELSYLHGHRNLIPGLEEALEGREEG EAFQAHVPAEKAYGAGSGGGGDYALQGGGGGSSGKDLDFQVEVVKVREAT PEELLHGHAHGGGSGGGSGGGSGGGSGGGSGGGIEGRMSLASGKSLLHEV DKDISQLTQAIVKNHKNLLKIAQYAAQNRRGLDLLFWEQGGLAKALQEQA AFLNITNSHVSILQERPPLENRVLTGWGLNWDLGLSQWAREALQTGGHHH HHHHH TtSlyD-Xa-gp21-8H: (SEQIDNO:61) MKVGQDKVVTIRYTLQVEGEVLDQGELSYLHGHRNLIPGLEEALEGREEG EAFQAHVPAEKAYGPHDPEGVQVVPLSAFPEDAEVVPGAQFYAQDMEGNP MPLTVVAVEGEEVTVDFNHPLAGKDLDFQVEVVKVREATPEELLHGHAHG GGSGGGSGGGSGGGSGGGSGGGIEGRMSLASGKSLLHEVDKDISQLTQAI VKNHKNLLKIAQYAAQNRRGLDLLFWEQGGLAKALQEQAAFLNITNSHVS ILQERPPLENRVLTGWGLNWDLGLSQWAREALQTGGHHHHHHHH TtSlyKQD-SlpA-Xa-gp21-8H: (SEQIDNO:62) MKVGQDKVVTIRYTLQVEGEVLDQGELSYLHGHRNLIPGLEEALEGREEG EAFQAHVPAEKAYGAGSGGGGYRYRQGGGGGSSGKDLDFQVEVVKVREAT PEELLHGHAHGGGSGGGSGGGSGGGSGGGSGGGMSESVQSNSAVLVHFTL KLDDGTTAESTRNNGKPALFRLGDASLSEGLEQHLLGLKVGDKTTFSLEP DAAFGVPSPDLIQYFSRREFMDAGEPEIGAIMLFTAMDGSEMPGVIREIN GDSITVDFNHPLAGQTVHFDIEVLEIDPALEAGGGSGGGSGGGSGGGSGG GSGGGIEGRMSLASGKSLLHEVDKDISQLTQAIVKNHKNLLKIAQYAAQN RRGLDLLFWEQGGLAKALQEQAAFLNITNSHVSILQERPPLENRVLTGWG LNWDLGLSQWAREALQTGGHHHHHHHH
[0114] The 8X-histidine-tagged proteins were produced in E. coli B121 Tuner and purified by standard Ni Sepharose-based immobilized metal ion affinity and size exclusion chromatography (HisTrap, Superdex 200; GE Healthcare).
[0115] Labeled peptides were chemically synthesized to have (in order from N-terminus to C-terminus) a Z- group (i.e., a carboxybenzyl group), a transglutaminase lysine donor sequence (K-tag), polyethylene glycol (PEG), peptide, and a Biotin label or Bipyrimidine Ruthenium (BPRu) complex. The primary chemical structures of the labeled peptides were:
[0116] KalbTG K-tag-Bi (Bi represents a Biotin label):
[0117] Z-RYESKG-PEG27-K(Bi)OH (2532.0 g/mol)
[0118] KalbTG K-tag-Ru (Ru represents a Ruthenium-based label for electrochemoluminescence): Z-RYESKG-PEG27-K(BPRu)-OH (2958.4 g/mol)
[0119] If not noted otherwise, typical labeling reactions were performed for 15 minutes at 37 C. in the presence of 65 M substrate protein, 1000 M label peptide and 0.1 M transglutaminase in 200 mM MOPS pH 7.4 for labeling with the KalbTG enzyme and at 37 C. in the presence of 72 M substrate protein, 720 M label peptide and 1 M transglutaminase in 200 mM MOPS pH 7.4 with the MTG enzyme. Transglutaminase labeling is described in more detail in applications US 2016/0178627 System and Method for Identification and Characterization of Transglutaminase Species and WO 2016/096785 IDENTIFICATION OF TRANSGLUTAMINASE SUBSTRATES AND USES THEREFOR.
[0120] The labeling reaction mix was separated by size exclusion chromatography (Superdex 200; GE Healthcare) and fractions containing labeled fusion proteins were isolated for subsequent analysis.
Analysis of Biotin- and Ruthenium Labeling and Immunoassay
[0121] The quality and quantity of labeling reactions were analyzed by SDS-PAGE. Efficiency of Biotin labeling was estimated by the shift in molecular weight of the bands in the coomassie-stained gel. Efficiency of Ruthenium labeling was estimated by the shift in molecular weight of the bands in the coomassie-stained gel and by in-gel fluorescence (BioRad ChemiDoc gel documentation system, Cy3 LED and filter set). Diagnostic immunoassays (Roche HTLV I/II) were performed according to the manufacturer's instructions on an Elecsys e411 instrument, replacing the biotin labeled antigen moiety in reagent R1 of the test by TtSlyD-Xa-gp21-8H biotin labeled with MTG or TtSlyKQD-SlpA-Xa-gp21-8H biotin labeled with KalbTG, respectively, and replacing the Ruthenium labeled antigen moiety in reagent R2 of the test by TtSlyD-Xa-gp21-8H Ruthenium labeled with MTG or TtSlyKQD-SlpA-Xa-gp21-8H Ruthenium labeled with KalbTG, respectively. Postive controls were performed with HTLV positive sera and the commercially available HTLV I/II kit. Negative controls were performed with HTLV negative sera and with HTLV positive sera in combination with different calibrator and control solutions.
[0122] For results, see
Mass Spectrometric Analysis of TtSlyQD-Xa-gp21-8H Ruthenium Labeling
[0123]
[0124] Two pooled fractions of the Ruthenium labelled fusion protein were analyzed, [1] TtSlyQD-Xagp21-8H_Fraction 17-19 and [2] TtSlyQDXa-gp21-8H_Fraction 20-23; unlabeled TtSlyQD-Xagp21-8H protein served a control (TtSlyQD-Xa-gp21-8H_Control).
[0125] Peptide mapping was performed. After peptide mapping by digestion with Trypsin, chromatographic separation, subsequent MS/MS analysis, and database searching in combination with manual interpretation tools, the following was found:
[0126] (a) The tryptic peptide 62-85 AYGAGSGGGGDYALQGGGGGSSGK SEQ ID NO:63 (numbering is referred to the sequence given below) with one label was found in both fractions TtSlyQD-Xa-gp21-8H_Fraction 17-19 and TtSlyQD-Xa-gp21-8H_Fraction 20-23.
[0127] (b) The tryptic peptide 195-222 ALQEQAAFLNITNSHVSILQERPPLENR SEQ ID NO:64 with one label was found in traces only in TtSlyQD-Xa-gp21-8H_Fraction 17-19.
[0128] (c) The tryptic peptide 241-255 EALQTGGHHHHHHHH SEQ ID NO:65 (His-Tag) with one label was found in both fractions TtSlyQD-Xa-gp21-8H_Fraction 17-19 and TtSlyQD-Xagp21-8H_Fraction 20-23.
[0129] (d) In addition to the above, also other Ruthenium labelled peptides/byproducts were found to be present in both Fractions, however in insignificant quantities.
[0130] Amino acid sequence of TtSlyQD-Xa-gp21-8H, tryptic peptides as given above in (a-c) are marked in boldface and underlined:
TABLE-US-00015 (SEQIDNO:60) MKVGQDKVVTIRYTLQVEGEVLDQGELSYLHGHRNLIPGLEEALEGREEG EAFQAHVPAEKAYGAGSGGGGDYALQGGGGGSSGKDLDFQVEVVKVREAT PEELLHGHAHGGGSGGGSGGGSGGGSGGGSGGGIEGRMSLASGKSLLHEV DKDISQLTQAIVKNHKNLLKIAQYAAQNRRGLDLLFWEQGGLAKALQEQA AFLNITNSHVSILQERPPLENRVLTGWGLNWDLGLSQWAREALQTGGHHH HHHHH
[0131] Recombinant gp21 (HTLV) antigen, site-specifically labeled with Biotin and Ruthenium using the bacterial transglutaminase of Kutzneria albida demonstrates the advantages of the invention in the Elecsys HTLV-I/II in-vitro diagnostic assay.
[0132] The results are presented in
[0133]
[0134]
[0135]
[0136] The amino acid sequence of TtSlyKQD-SlpA-Xa-gp21-8H is as follows:
TABLE-US-00016 (SEQIDNO:62) MKVGQDKVVTIRYTLQVEGEVLDQGELSYLHGHRNLIPGLEEALEGREEG EAFQAHVPAEKAYGAGSGGGGYRYRQGGGGGSSGKDLDFQVEVVKVREAT PEELLHGHAHGGGSGGGSGGGSGGGSGGGSGGGMSESVQSNSAVLVHFTL KLDDGTTAESTRNNGKPALFRLGDASLSEGLEQHLLGLKVGDKTTFSLEP DAAFGVPSPDLIQYFSRREFMDAGEPEIGAIMLFTAMDGSEMPGVIREIN GDSITVDFNHPLAGQTVHFDIEVLEIDPALEAGGGSGGGSGGGSGGGSGG GSGGGIEGRMSLASGKSLLHEVDKDISQLTQAIVKNHKNLLKIAQYAAQN RRGLDLLFWEQGGLAKALQEQAAFLNITNSHVSILQERPPLENRVLTGWG LNWDLGLSQWAREALQTGGHHHHHHHH.