Soluble Sortase A

Abstract

Herein is reported a polypeptide comprising the amino acid sequence of SEQ ID NO: 38 as sole Listeria monocytogenes derived polypeptide and its use in conjugating polypeptides.

Claims

1. A non-native polypeptide consisting of SEQ ID NO: 38.

2. A method for conjugating two polypeptides comprising the step of incubating in an aqueous environment the polypeptide according to claim 1 with a first polypeptide comprising a sortase-motif comprising the amino acid sequence LPXTG (SEQ ID NO: 01, wherein X can be any amino acid residue) or LPXTA (SEQ ID NO: 41, wherein X can be any amino acid residue), and a second polypeptide comprising an oligoglycine or oligoalanine or a cysteine amino acid residue followed by one to three glycine or alanine amino acid residues at its N-terminus.

3. A method for enzymatically producing a polypeptide comprising the following step incubating in an aqueous environment i) a first polypeptide comprising the amino acid sequence LPXTG (SEQ ID NO: 01, wherein X can be any amino acid residue) or LPXTA (SEQ ID NO: 41, wherein X can be any amino acid residue), ii) a second polypeptide that comprises i) a glycinyl, an alaninyl, or a cysteinyl compound at its N-terminus, or ii) an oligoglycine, or oligoalanine, or a cysteine amino acid residue followed by one to three glycine or alanine amino acid residues at its N-terminus, or iii) a lysine amino acid residue within its 5 N-terminal amino acid residues, and iii) the polypeptide according to claim 1; thereby producing a polypeptide.

4. The method according to claim 3, wherein the second polypeptide has at its N-terminus the amino acid sequence GGG, AAA, CGG, CAA, KGG or KAA.

5. The method according to claim 3, 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) or LPXTA (SEQ ID NO: 41, wherein X can be any amino acid residue).

6. The method according to claim 3 wherein the first polypeptide comprises at its C-terminus the amino acid sequence LPETG (SEQ ID NO: 04) or LPETA (SEQ ID NO: 42).

7. The method according to claim 3, wherein the first polypeptide and the second polypeptide are independently of each other selected from an antibody variable domain, an antibody heavy chain Fab-fragment, an antibody Fc-region, a tag, and a peptide comprising the amino acid sequence LPXTG (SEQ ID NO: 01, wherein X can be any amino acid residue) or LPXTA (SEQ ID NO: 41, wherein X can be any amino acid residue), a linker and a non-sortase motif moiety.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1 Different concentrations of Listeria monocytogenes Sortase A variant C (triangles) and St.py. SrtA (crosses) were incubated for 2h with 92 μM glucose Dehydrogenase harboring a LPKTA motive and 7 μM AAAA-biotin and 7 M GGGG-biotin. After incubation on the Streptavidin beads, 10 μl of the suspension where analyzed for the reporter enzyme activity.

(2) FIG. 2 0.67 mg/ml of Listeria monocytogenes SrtA variant C (diamond) and St.py. SrtA (square) were incubated for different hours with 92 μM glucose Dehydrogenase harboring a LPKTA motive and 7 μM AAAA-biotin and 7 μM GGGG-biotin. After incubation on the Streptavidin beads, 10 μl of the suspension where analyzed for the reporter enzyme activity.

(3) FIG. 3 1 mg/ml of Listeria monocytogenes SrtA variant C was incubated for 2 h with different concentrations of glucose Dehydrogenase harboring a LPKTA motive and 14 μM GGGG-Biotin. After incubation on the Streptavidin beads 40 μl of the suspension where analyzed for the reporter enzyme activity.

(4) FIG. 4 3 mg/ml of St.py. SrtA was incubated for 2 h with different concentrations of Glucose Dehydrogenase harboring a LPKTA motive and 14 μM GGGG-Biotin. After incubation on the streptavidin beads, 40 μl of the suspension where analyzed for the reporter enzyme activity.

(5) 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

(6) Recombinant DNA Techniques

(7) 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.

(8) Gene and Oligonucleotide Synthesis

(9) 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).

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

(11) 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).

(12) 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

(13) 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

(14) Generation of an Expression Plasmid for Soluble Sortase A Staphylococcus aureus derived Sortase A The sortase gene encodes an N-terminally truncated Staphylococcus aureus sortase A (60-206) molecule (amino acid sequence of SEQ ID NO: 05).

(15) 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 LacI gene to allow induction of transcription using IPTG.

(16) 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.

(17) 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.

(18) 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).

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

(20) TABLE-US-00019 (SEQ ID NO: 05) QAKPQIPKDKSKVAGYIEIPDADIKEPVYPGPATPEQLNRGVSFAEENES LDDQNISIAGHTFIDRPNYQFTNLKAAKKGSMVYFKVGNETRKYKMTSIR DVKPTDVGVLDEQKGKDKQLTLITCDDYNEKTGVWEKRKIFVATEVK.

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

(22) Streptococcus pyogenes Derived Sortase A

(23) The sortase gene encodes an N-terminally truncated Streptococcus pyogenes sortase A molecule (amino acid sequence of SEQ ID NO: 06).

(24) 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 LacI gene to allow induction of transcription using IPTG.

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

(26) 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.

(27) 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. pyogenes sortase A encoding nucleic acid, and the bovine growth hormone polyadenylation sequence (BGH pA).

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

(29) TABLE-US-00020 (SEQ ID NO: 06) VLQAQMAAQQLPVIGGIAIPELGINLPIFKGLGNTELIYGAGTMKEEQVM GGENNYSLASHHIFGITGSSQMLFSPLERAQNGMSIYLTDKEKIYEYIIK DVFTVAPERVDVIDDTAGLKEVTLVTCTDIEATERIIVKGELKTEYDFDK APADVLKAFNHSYNQVST.

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

(31) Listeria monocytogenes Derived Sortase A

(32) Different sortase genes encoding N-terminally truncated Listeria monocytogenes sortase A molecules were expressed:

(33) A: Listeria monocytogenes Sortase A variant A as reported herein:

(34) TABLE-US-00021 (SEQ ID NO: 36) EKDATFDFESVQLPSMTSVIKGAANYDKDAVVGSIAVPSVDVNLLVFKGT NTANLLAGATTMRSDQVMGKGNYPLAGHHMRDESMLFGPIMKVKKGDK IYLTDLENLYEYTVTETKTIDETEVSVIDDTKDARITLITCDKPTETTKR FVAVGELEKTEKLTKELENKYFPSK

(35) B: Listeria monocytogenes Sortase A variant B as reported herein:

(36) TABLE-US-00022 (SEQ ID NO: 37) SVIKGAANYDKDAVVGSIAVPSVDVNLLVFKGTNTANLLAGATTMRSDQ VMGKGNYPLAGHHMRDESMLFGPIMKVKKGDKIYLTDLENLYEYTVTET KTIDETEVSVIDDTKDARITLITCDKPTETTKRFVAVGELEKTEKLTKEL ENKYFPSK

(37) C: Listeria monocytogenes Sortase A variant C as reported herein:

(38) TABLE-US-00023 (SEQ ID NO: 38) ANYDKDAVVGSIAVPSVDVNLLVFKGTNTANLLAGATTMRSDQVMGKG NYPLAGHHMRDESMLFGPIMKVKKGDKIYLTDLENLYEYTVTETKTIDET EVSVIDDTKDARITLITCDKPTETTKRFVAVGELEKTEKLTKELENKYFP SK

(39) D: Listeria monocytogenes Sortase A variant D as reported herein:

(40) TABLE-US-00024 (SEQ ID NO: 39) ANYDKDAVVGSIAVPSVDVNLLVFKGTNTANLLAGATTMRSDQVMGKG NYPLAGHHMRDESMLFGPIMKVKKGDKIYLTDLENLYEYTVTETKTIDET EVSVIDDTKDARITLITCDKPTETTKRFVAVGELEKTEK

(41) E: Listeria monocytogenes Sortase A variant E as reported herein:

(42) TABLE-US-00025 (SEQ ID NO: 40) GSIAVPSVDVNLLVFKGTNTANLLAGATTMRSDQVMGKGNYPLAGHHMR DESMLFGPIMKVKKGDKIYLTDLENLYEYTVTETKTIDETEVSVIDDTKD ARITLITCDKPTETTKRFVAVGELEKTEKLTKELENKYFPSK

(43) The expression plasmid for the expression of the truncated sortases in E. coli cells comprised besides the 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 LacI gene to allow induction of transcription using IPTG.

(44) The transcription unit of the soluble sortase comprised the following functional elements: a T5 promoter, a purification tag, the Listeria monocytogenes sortase A variant encoding nucleic acid, and the To and fd termination sequences.

(45) The expression plasmid for the transient expression of truncated sortases in HEK293 cells comprised besides the 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.

(46) 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, the L. monocytogenes sortase A variant encoding nucleic acid, and the bovine growth hormone polyadenylation sequence (BGH pA).

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

Example 2

(48) Transient Expression and Analytical Characterization

(49) E. coli:

(50) 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.

(51) 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.

(52) HEK:

(53) 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.).

(54) 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.

(55) 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.

(56) The Listeria monocytogenes Sortase A variant D showed very low expression yields.

Example 3

(57) Activity Assay

(58) Forty microliters of each variant were mixed with 80 μl substrate solution (50 mM Tris pH 7.5, 200 mM NaCl, 10 mM CaCl.sub.2, 30 μM of a glucose dehydrogenase (containing one of the substrates of the Sortase reaction (LPXTG)) and biotin (containing the other substrate of the Sortase reaction, GGGG)). This reaction mixture was incubated at 37° C. for 2 hours. Thereafter the 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. 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 10 μL thereof are transferred to a new well. Thereto 150 μL test buffer (0.2 M sodium citrate, pH 5.8, 0.3 g/L 4-nitrosoanilin, 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.

(59) The activity of the different variants is shown in the following Table.

(60) TABLE-US-00026 Listeria monocytogenes sortase variant maximum dE/min A (SEQ ID NO: 36 + C-terminal SEQ ID NO: 32) 0.037 B (SEQ ID NO: 37 + C-terminal SEQ ID NO: 32) 0.026 C (SEQ ID NO: 38 + C-terminal SEQ ID NO: 32) 0.113 D (SEQ ID NO: 39 + C-terminal SEQ ID NO: 32) 0.044 E (SEQ ID NO: 40 + C-terminal SEQ ID NO: 32) 0.019

Example 4

(61) Kinetic Assays

(62) Enzyme Amount

(63) Different concentrations of Sortase (0.02 mg/ml, 0.17 mg/ml, 0.33 mg/ml, 0.67 mg/ml, 1.33 mg/ml, 2.00 mg/ml, 2.67 mg/ml, 3.33 mg/ml) were mixed with substrate solution (50 mM Tris pH 7.5, 200 mM NaCl, 10 mM CaCl.sub.2, 92 μM glucose dehydrogenase (containing one of the substrates of the Sortase reaction (LPKTA) and 7 μM biotin (containing the other substrate of the Sortase reaction, AAAA) and 7 μM biotin (containing the other substrate of the Sortase reaction, GGGG)). This reaction mixture was incubated at 37° C. for 2 hours. The reaction was stopped by addition of a 20-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. 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 multi-well plates using a magnet and a vacuum pump. Afterwards the beads were resuspended in 100 μL citrate buffer and 10 μL thereof are transferred to a new well. Thereto 150 μL test buffer (0.2 M sodium citrate, pH 5.8, 0.3 g/L 4-nitrosoanilin, 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.

(64) The activity of the St.py. SrtA and Listeria monocytogenes Sortase A variant C are shown in FIG. 1.

(65) Incubation Time

(66) A fixed concentrations of Sortase (0.67 mg/ml) were mixed with substrate solution (50 mM Tris pH 7.5, 200 mM NaCl, 10 mM CaCl.sub.2, 92 μM glucose dehydrogenase (containing one of the substrates of the Sortase reaction (LPKTA) and 7 μM biotin (containing the other substrate of the Sortase reaction, AAAA) and 7 μM biotin (containing the other substrate of the Sortase reaction, GGGG)). This reaction mixture was incubated at 37° C. for 2, 6.5 or 18 hours. The reaction was stopped by addition of a 20-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. 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 multi-well plates using a magnet and a vacuum pump. Afterwards the beads were resuspended in 100 μL citrate buffer and 10 μL thereof are transferred to a new well. Thereto 150 μL test buffer (0.2 M sodium citrate, pH 5.8, 0.3 g/L 4-nitrosoanilin, 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.

(67) The activity of the St.py. SrtA and Listeria monocytogenes Sortase A variant C are shown in FIG. 2.

(68) Sortase-Motif Polypeptide Concentration

(69) A fixed concentrations of Listeria monocytogenes Sortase variant C (1 mg/ml) were mixed with substrate solution (50 mM Tris pH 7.5, 200 mM NaCl, 10 mM CaCl.sub.2, 2.5, 5, 10, 20, 40, 80, 160, 325, 650, 1300 μM glucose dehydrogenase (containing one of the substrates of the Sortase reaction (LPKTA) and 14 μM biotin (containing the other substrate of the Sortase reaction, GGGG)). This reaction mixture was incubated at 37° C. for 2 hours. The reaction was stopped by addition of a 20-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. 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 multi-well plates using a magnet and a vacuum pump. Afterwards the beads were resuspended in 100 μL citrate buffer and 40 μL thereof are transferred to a new well. Thereto 150 μL test buffer (0.2 M sodium citrate, pH 5.8, 0.3 g/L 4-nitrosoanilin, 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.

(70) The activity of the Listeria monocytogenes Sortase A variant C is shown in FIG. 3.

(71) Sortase-Motif Polypeptide Concentration

(72) A fixed concentrations of Staphylococcus pyogenes Sortase (3 mg/ml) were mixed with substrate solution (50 mM Tris pH 7.5, 200 mM NaCl, 10 mM CaCl.sub.2, 2.5, 5, 10, 20, 40, 80, 160, 325, 650, 1300 μM glucose dehydrogenase (containing one of the substrates of the Sortase reaction (LPKTA) and 14 μM biotin (containing the other substrate of the Sortase reaction, GGGG)). This reaction mixture was incubated at 37° C. for 2 hours. The reaction was stopped by addition of a 20-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. 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 multi-well plates using a magnet and a vacuum pump. Afterwards the beads were resuspended in 100 μL citrate buffer and 40 μL thereof are transferred to a new well. Thereto 150 μL test buffer (0.2 M sodium citrate, pH 5.8, 0.3 g/L 4-nitrosoanilin, 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.

(73) The activity of the Streptococcus pyogenes Sortase A is shown in FIG. 4.

(74) 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.