Production of thioesters using sortase

Abstract

Herein is reported a method for the enzymatic production of a thioester comprising incubating i) a first polypeptide comprising the amino acid sequence LPXTG (SEQ ID NO: 01, wherein X can be any amino acid residue), ii) a second polypeptide that has at its N-terminus a cysteine amino acid residue or is a cysteinyl compound, and iii) a third polypeptide with sortase A activity.

Claims

1. A method for enzymatic conjugation of two polypeptides comprising: incubating in an aqueous environment a first polypeptide comprising the amino acid sequence LPXTG (SEQ ID NO: 01, wherein X can be any amino acid residue); a second polypeptide that has at its N-terminus a cysteine amino acid residue followed by one to three glycine amino acid residues; and a third polypeptide that is a sortase A or a catalytically active fragment thereof; thereby forming a thioester bond.

2. The method according to claim 1, wherein the third polypeptide is selected from Staphylococcus aureus sortase A, a catalytically active fragment of Staphylococcus aureus sortase A, Listeria monocytogenes Sortase A, and a catalytically active fragment of Listeria monocytogenes Sortase A.

3. The method according to claim 1, wherein the incubating is further in the presence of a thiol additive.

4. The method according to claim 3, wherein the thiol additive is selected from the group consisting of thiophenol, 4-mercaptophenylacetic acid (MPAA), and 2-mercaptoethanesulfonate (MESNA), and combinations thereof.

5. The method according to claim 1, wherein the first polypeptide comprises at its C-terminus the amino acid sequence LPXTG (SEQ ID NO: 01, wherein X can be any amino acid residue).

6. The method according to claim 1, wherein the first polypeptide comprises at its C-terminus the amino acid sequence LPETG (SEQ ID NO: 04).

7. The method according to claim 1, wherein the second polypeptide has the amino acid sequence CGGG (SEQ ID NO: 02) at its N-terminus.

8. The method according to claim 1, wherein the first polypeptide and the second polypeptide are independently of each other selected from the group consisting of an antibody variable domain, an antibody heavy chain, an antibody Fab-fragment, an antibody Fc-region, a tag, and a linker.

9. The method according to claim 1, wherein the third polypeptide has the amino acid sequence of SEQ ID NO: 05 or SEQ ID NO: 06.

10. A method for enzymatic conjugation of two polypeptides comprising: incubating in an aqueous environment a first polypeptide comprising the amino acid sequence LPXTG (SEQ ID NO: 01, wherein X can be any amino acid residue); a second polypeptide that has at its N-terminus a cysteine amino acid residue followed by one to three glycine amino acid residues or one to three alanine amino acid residues; and a third polypeptide that is Listeria monocytogenes Sortase A or a catalytically active fragment of Listeria monocytogenes Sortase A that is not a catalytically active fragment derived from Staphylococcus aureus sortase A, thereby forming a thioester bond.

11. The method according to claim 10, wherein the incubating is further in the presence of a thiol additive.

12. The method according to claim 11, wherein the thiol additive is selected from the group consisting of thiophenol, 4-mercaptophenylacetic acid (MPAA), and 2-mercaptoethanesulfonate (MESNA), and combinations thereof.

13. The method according to claim 10, wherein the first polypeptide comprises at its C-terminus the amino acid sequence LPXTG (SEQ ID NO: 01, wherein X can be any amino acid residue).

14. The method according to claim 10, wherein the first polypeptide comprises at its C-terminus the amino acid sequence LPETG (SEQ ID NO: 04).

15. The method according to claim 10, wherein the second polypeptide has the amino acid sequence CGGG (SEQ ID NO: 02) or CAAA (SEQ ID NO: 03) at its N-terminus.

16. The method according to claim 10, wherein the first polypeptide and the second polypeptide are independently of each other selected from the group consisting of an antibody variable domain, an antibody heavy chain, an antibody Fab-fragment, an antibody Fc-region, a tag, and a linker.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1 Scheme showing the native chemical ligation reaction.

(2) FIG. 2 Scheme showing the use of Sortase in NCL.

(3) FIG. 3 Chromatogram of the reaction product of Example 3.

(4) FIG. 4A Mass-spectrum of peak 1 of the chromatogram of FIG. 3.

(5) FIG. 4B Mass-spectrum of peak 2 of the chromatogram of FIG. 3.

(6) FIG. 4C Mass-spectrum of peak 3 of the chromatogram of FIG. 3.

(7) FIG. 4D Mass-spectrum of peak 4 of the chromatogram of FIG. 3.

(8) FIG. 4E Mass-spectrum of peak 5 of the chromatogram of FIG. 3.

(9) FIG. 4F Mass-spectrum of peak 6 of the chromatogram of FIG. 3.

(10) FIG. 4G Mass-spectrum of peak 7 of the chromatogram of FIG. 3.

(11) FIG. 4H Mass-spectrum of peak 8 of the chromatogram of FIG. 3.

(12) FIG. 5 Enzymatic activity of Sa-SrtA using a REIA with different Nucleophiles.

(13) FIG. 6 Enzymatic activity of Lm-SrtA using a REIA with different Nucleophiles.

(14) The following examples, figures and sequences are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit of the invention.

EXAMPLES

(15) Recombinant DNA Techniques

(16) Standard methods were used to manipulate DNA as described in Sambrook, J. et al., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989. The molecular biological reagents were used according to the manufacturer's instructions.

(17) Gene and Oligonucleotide Synthesis

(18) Desired gene segments were prepared by chemical synthesis at Geneart GmbH (Regensburg, Germany). The synthesized gene fragments were cloned into an E. coli plasmid for propagation/amplification. The DNA sequences of subcloned gene fragments were verified by DNA sequencing. Alternatively, short synthetic DNA fragments were assembled by annealing chemically synthesized oligonucleotides or via PCR. The respective oligonucleotides were prepared by metabion GmbH (Planegg-Martinsried, Germany).

(19) Description of the Basic/Standard Mammalian Expression Plasmid

(20) For the expression of a desired gene/protein (e.g. full length antibody heavy chain, full length antibody light chain, or an Fc-chain containing an oligoglycine at its N-terminus) a transcription unit comprising the following functional elements is used: the immediate early enhancer and promoter from the human cytomegalovirus (P-CMV) including intron A, a human heavy chain immunoglobulin 5′-untranslated region (5′UTR), a murine immunoglobulin heavy chain signal sequence, a gene/protein to be expressed (e.g. full length antibody heavy chain), and the bovine growth hormone polyadenylation sequence (BGH pA).

(21) Beside the expression unit/cassette including the desired gene to be expressed the basic/standard mammalian expression plasmid contains an origin of replication from the vector pUC18 which allows replication of this plasmid in E. coli, and a beta-lactamase gene which confers ampicillin resistance in E. coli.
Protein Determination

(22) The protein concentration of purified polypeptides was determined by determining the optical density (OD) at 280 nm, using the molar extinction coefficient calculated on the basis of the amino acid sequence of the polypeptide.

Example 1

(23) Generation of an Expression Plasmid for Soluble S. aureus Sortase A

(24) The sortase gene encodes an N-terminally truncated Staphylococcus aureus sortase A (60-206) molecule (amino acid sequence of SEQ ID NO: 05).

(25) The expression plasmid for the expression of soluble sortase in E. coli cells comprised besides the soluble sortase expression cassette an origin of replication from the vector pUC18, which allows replication of this plasmid in E. coli, and the URA3 gene as selectable marker, and the Lad gene to allow induction of transcription using IPTG.

(26) The transcription unit of the soluble sortase comprised the following functional elements: a T5 promoter, a purification tag, an N-terminally truncated S. aureus sortase A encoding nucleic acid, and the To and fd termination sequences.

(27) The expression plasmid for the transient expression of soluble sortase in HEK293 cells comprised besides the soluble sortase expression cassette an origin of replication from the vector pUC18, which allows replication of this plasmid in E. coli, and a beta-lactamase gene which confers ampicillin resistance in E. coli.

(28) The transcription unit of the soluble sortase comprised the following functional elements: the immediate early enhancer and promoter from the human cytomegalovirus (P-CMV) including intron A, a human heavy chain immunoglobulin 5′-untranslated region (5′UTR), a murine immunoglobulin heavy chain signal sequence, a purification tag encoding nucleic acid, an N-terminally truncated S. aureus sortase A encoding nucleic acid, and the bovine growth hormone polyadenylation sequence (BGH pA).

(29) The amino acid sequence of the mature soluble sortase is

(30) TABLE-US-00002 (SEQ ID NO: 05) QAKPQIPKDKSKVAGYIEIPDADIKEPVYPGPATPEQLNRGVSFAEENES LDDQNISIAGHTFIDRPNYQFTNLKAAKKGSMVYFKVGNETRKYKMTSIR DVKPTDVGVLDEQKGKDKQLTLITCDDYNEKTGVWEKRKIFVATEVK.

(31) The purification tag has the amino acid sequence MRGSHHHHHHGS (SEQ ID NO: 32).

Example 2

(32) Transient Expression and Analytical Characterization

(33) E. coli:

(34) The recombinant production of Sortase was performed by growing E. coli cells transformed with the respective Sortase expression plasmids to an OD578 of approx. 0.9 at 37° C. (pre-culture). At this OD578 of approx. 0.9 protein expression was induced by adding 2 mM IPTG and growing the cells for an additional 24 hours at 28° C. Thereafter, cells were harvested by centrifugation and lysed via high pressure using a homogenizer. Cell lysates were centrifuged to remove cell debris and subsequently the cell lysates were stored at reduced temperature (e.g. −80° C.) until purification. Soluble Sortase was purified using Ni-NTA chromatography followed by size exclusion chromatography. For depletion of endotoxins an anion exchange chromatography was performed in flow through mode. The protein concentration of sortase preparations was determined by measuring the optical density (OD) at 280 nm, using the molar extinction coefficient calculated on the basis of the amino acid sequence. Purity and integrity of sortase was determined by SDS-PAGE in the presence and absence of a reducing agent (5 mM 1,4-dithiotreitol) and staining with Coomassie brilliant blue.

(35) The protein concentration was determined by measuring the optical density (OD) at 280 nm, using the molar extinction coefficient calculated on the basis of the amino acid sequence. Purity was analyzed by SDS-PAGE in the presence and absence of a reducing agent (5 mM 1,4-dithiotreitol) and staining with Coomassie brilliant blue.

(36) HEK:

(37) The recombinant production was performed by transient transfection of HEK293 cells (human embryonic kidney cell line 293-derived) cultivated in F17 Medium (Invitrogen Corp.). For transfection “293-Fectin” Transfection Reagent (Invitrogen) was used. Transfection was performed as specified in the manufacturer's instructions. Cell culture supernatants were harvested three to seven (3-7) days after transfection. Supernatants were stored at reduced temperature (e.g. −80° C.).

(38) General information regarding the recombinant expression of human immunoglobulins in e.g. HEK293 cells is given in: Meissner, P. et al., Biotechnol. Bioeng. 75 (2001) 197-203.

(39) The protein concentration was determined by measuring the optical density (OD) at 280 nm, using the molar extinction coefficient calculated on the basis of the amino acid sequence. Purity was analyzed by SDS-PAGE in the presence and absence of a reducing agent (5 mM 1,4-dithiotreitol) and staining with Coomassie brilliant blue.

Example 3

(40) Sortase Mediated Conjugation

(41) A reaction mixture comprising 0.5 mM of the polypeptide LCR640-ULPETGGGRRC (U: LCR640 fluorophore conjugated beta-alanine; SEQ ID NO: 33) Fc-region fragment comprising a LPETG sortase motif (SEQ ID NO: 04), 1.5 mM of an N-terminal biotinylated N-terminal cysteine comprising peptide with the C-terminally biotinylated amino acid sequence CAAA (SEQ ID NO: 03) and 50 μM Staphylococcus aureus Sortase A in 50 mM Tris pH 7.5, 150 mM NaCl, 10 mM CaCl.sub.2 was incubated at 37° C. for 18 hours.

(42) In the samples were analyzed without stopping the reaction.

(43) The samples (10 μl) were injected on a Vydac C18 column of an LC-Ms system and separated with a 30 min. linear gradient to 100% buffer B (buffer A (v/v): 95% water, 5% acetonitrile, 0.1% trifluoro acetic acid (TFA); buffer B (v/v): 5% water, 95% Acetonitrile, 0.1% TFA). The respective chromatogram is shown in FIG. 3.

(44) The Analysis of the reaction mixture with LC-ESI-TOF-MS in positive ion mode shows in peak 4 the product of the native chemical ligation reaction with the mass of 2155 Da.

(45) The respective fragment pattern and masses are shown in the following Table.

(46) TABLE-US-00003 LCR-----U-------L-------P-----E----T-------G----G----G----R-----R---C-NH 1840 726 1114 1237 (1254) 603 726 512 603  1 Educt: LPETG 1840  2 LCR-GGRR 1329  3 Educt: LPETG dimer 3676  4 Educt: G-Bio 830  5 Educt: C-Bio 919  6 LCR-LPETG-Bio 2067  7 LCR-LPETC-Bio 2156

Example 4

(47) Reporter Immobilization Assay

(48) More detailed analysis of Sortases generating a thioester for native chemical ligation was done using a reporter immobilization assay (REIA) as reported in European Patent application EP14198535 and as outlined below.

(49) Reaction Mixture:

(50) 20 μM Staphylococcus aureus sortase A (Sa-SrtA) 100 μM nucleophile (GGGG/AAAA/CAAA) 20 μM glucose dehydrogenase with C-terminal sortase motif (LPXTG) 250 mM MESNA 0.5 mM TCEP or 100 μM Listeria monocytogenes sortase A (Lm-SrtA) 100 μM nucleophile (GGGG/AAAA/CAAA) 20 μM glucose dehydrogenase with C-terminal sortase motif (LPXTG) 250 mM MESNA.

(51) The glucose dehydrogenase was expressed and purified as described in WO 2007/118647.

(52) Both reaction mixtures were prepared in 50 mM Tris-HCl buffer pH 7.5, 150 mM NaCl, 10 mM CaCl.sub.2.

(53) The reaction mixture was incubated at 37° C. for up to 60 hours. The reaction was stopped by addition of a 60-fold excess of inhibition buffer (50 mM Tris, pH 7.5, 200 mM NaCl, 10 mM CaCl.sub.2, 5 mM iodoacetamide). The stopped reaction mixture was centrifuged for 10 min at 5000×g. The supernatant (50 μL) was added to 100 μL of 50 mM Tris buffer (pH 7.5) comprising 200 mM NaCl, 10 mM CaCl.sub.2 and streptavidin coated magnetic beads. The mixture was incubated for 30 min at 30° C. with shaking at 200 rpm. Thereafter the magnetic beads were washed five times with 300 μL washing buffer each (50 mM Tris, pH 7.5, 200 mM NaCl, 10 mM CaCl.sub.2, 5 mg/mL BSA, 0.1% Triton X-100) in V-bottom micro-titer-plates using a magnet and a vacuum pump. Afterwards the beads were resuspended in 100 μL citrate buffer and 80 μL thereof was transferred to a new well. Thereto 150 μL test buffer (0.2 M sodium citrate, pH 5.8, 0.3 g/L 4-nitrosoaniline, 1 mM CaCl.sub.2, 30 mM glucose) were added. The kinetic of the reporter enzyme was measured over a time period of 5 min at 620 nm.

(54) The results are shown in FIGS. 5 and 6.

(55) Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the descriptions and examples should not be construed as limiting the scope of the invention.