Transamidation reaction in deep eutectic solvents

Abstract

Herein is reported a method for the enzymatic production of a polypeptide comprising the step of incubating 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 has 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) a third polypeptide with sortase A activity, in a deep eutectic solvent and thereby producing a polypeptide.

Claims

1. A method for the enzymatic production of a polypeptide comprising incubating in a deep eutectic solvent i) a first polypeptide comprising the amino acid sequence LPXTA (SEQ ID NO: 41), wherein X can be any amino acid residue, a second polypeptide that has 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) Staphylococcus aureus sortase A or Listeria monocytogenes sortase A, wherein the deep eutectic solvent is a mixture of choline chloride with glycerol at a molar ratio of 1:2 comprising up to 5% (v/v) aqueous co-solvent; thereby producing a polypeptide.

2. The method according to claim 1, wherein the second polypeptide has at its N-terminus the amino acid sequence GGG, AAA, CGG, CAA, KGG, or KAA.

3. The method according to claim 1, wherein the first polypeptide comprises at its C-terminus the amino acid sequence LPXTA (SEQ ID NO: 41), wherein X can be any amino acid residue.

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

5. The method according to claim 1, 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, a linker, and a non-sortase motif moiety.

6. A method for the enzymatic production of a polypeptide comprising incubating in a deep eutectic solvent 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 has i) a glycinyl or a cysteinyl compound at its N-terminus, or ii) an oligoglycine or iii) a lysine amino acid residue within its 5 N-terminal amino acid residues, and iii) Staphylococcus aureus sortase A or Listeria monocytogenes sortase A, wherein the deep eutectic solvent is a mixture of choline chloride with glycerol at a molar ratio of 1:2 comprising up to 5% (v/v) aqueous co-solvent; thereby producing a polypeptide.

7. The method according to claim 6, wherein the second polypeptide has at its N-terminus the amino acid sequence GGG, KGG, or KAA.

8. The method according to claim 6, 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.

9. The method according to claim 8, wherein the first polypeptide comprises at its C-terminus the amino acid sequence LPETG (SEQ ID NO: 04) or LPETA (SEQ ID NO: 42).

10. The method according to claim 6, 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, a linker, and a non-sortase motif moiety.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1 Chromatogram of the reaction product of Example 3; 1: sortase; 2: GGGWW-BHQ2 (SEQ ID NO: 46); 3: LCR640-ULPETGGRRC (SEQ ID NO: 175)+B: GGGWW-BHQ2 (SEQ ID NO: 46); 4: LCR640-ULPETGGGWW-BHQ2 (SEQ ID NO: 176)

(2) FIG. 2 Mass-spectrum of the peak of the chromatogram of FIG. 1; A:1, B:2, C:3, D:4.

(3) FIG. 3 Chromatogram of the reaction product of Example 4.

(4) FIG. 4 Mass-spectrum of the peak of the chromatogram of FIG. 3; A:1, B:2, C:3, D:4, E:5, F:6, G:7, H:8.

(5) FIG. 5 Enzymatic activity of soluble St.au. SrtA using a REIA in different solvents; diamond: water; square: DES-1 (ChCl:G); triangle: DES-2 (ChCl:PE).

(6) FIG. 6 Enzymatic activity of soluble Listeria monocytogenes SrtA using a REIA in different solvents; diamond: water; square: DES-1 (ChCl:G); triangle: DES-2 (ChCl:PE).

(7) FIG. 7 Absolute reaction yields (area under peak) of sortase reaction in DES containing different amounts of aqueous co-solvent. Indicated mixtures of ChCl:Glycerol and water were prepared with 200 mM NaCl, 10 mM CaCl.sub.2 and 50 mM Tris/HCl pH 7.5 and 0.05 mM Sa-SrtA, 0.5 mM LCRed640-LPETGGRRC (SEQ ID NO: 177) and 5 mM GGGG-Peg-Biotin (SEQ ID NO: 30). Samples were taken at indicated time points and analyzed by HPLC. Error bars represent the standard deviation of three independent measurements.

(8) FIG. 8 Absolute reaction yields (area under peak) of sortase reactions in ChCl:Glycerol containing 25% and 100% water. Both solvents were prepared with 200 mM NaCl, 10 mM CaCl.sub.2 and 50 mM Tris/HCl pH 7.5, 15 μM Sa-SrtA, 0.15 mM LCRed640-LPETGGRRC (SEQ ID NO: 177) and 0.15 mM GGWWK-BHQ2 (SEQ ID NO: 178). The reaction was analyzed at indicated time points for the generation of ligation product. Error bars represent the standard deviation of three independent measurements.

(9) 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

(10) Recombinant DNA Techniques

(11) 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.

(12) Gene and Oligonucleotide Synthesis

(13) 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).

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

(15) 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. shortened Sortase A of Staphylococcus aureus), and the bovine growth hormone polyadenylation sequence (BGH pA).

(16) 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

(17) 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

(18) Generation of an Expression Plasmid for Sortase A

(19) Staphylococcus aureus Derived Sortase A

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

(21) The expression plasmid for the expression of sortase 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 Lad gene to allow induction of transcription using IPTG.

(22) The transcription unit of the 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.

(23) The expression plasmid for the transient expression of sortase 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.

(24) The transcription unit of the 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).

(25) The amino acid sequence of the mature sortase is

(26) TABLE-US-00011 (SEQ ID NO: 05) QAKPQIPKDKSKVAGYIEIPDADIKEPVYPGPATPEQLNRGVSFAEENES LDDQNISIAGHTFIDRPNYQFTNLKAAKKGSMVYFKVGNETRKYKMTSIR DVKPTDVGVLDEQKGKDKQLTLITCDDYNEKTGVWEKRKIFVATEVK.

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

(28) Streptococcus pyogenes Derived Sortase A

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

(30) The expression plasmid for the expression of sortase 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 Lad gene to allow induction of transcription using IPTG.

(31) The transcription unit of the 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.

(32) The expression plasmid for the transient expression of sortase 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.

(33) The transcription unit of the 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).

(34) The amino acid sequence of the mature sortase is

(35) TABLE-US-00012 (SEQ ID NO: 06) VLQAQMAAQQLPVIGGIAIPELGINLPIFKGLGNTELIVGAGTMKEEQVM GGENNYSLASHHIFGITGSSQMLFSPLERAQNGMSIYLTDKEKIYEYIIK DVFTVAPERVDVIDDTAGLKEVTLVTCTDIEATERIIVKGELKTEYDFDK APADVLKAFNHSYNQVST.

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

(37) Listeria monocytogenes Derived Sortase A

(38) The sortase gene encodes an N-terminally truncated Listeria monocytogenes sortase A (73-222) molecule (amino acid sequence of SEQ ID NO: 38).

(39) The expression plasmid for the expression of the truncated Listeria monocytogenes 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 Lad gene to allow induction of transcription using IPTG.

(40) The transcription unit of the 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.

(41) 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.

(42) The expression plasmid for the transient expression of sortase 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.

(43) The transcription unit of the 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 L. monocytogenes sortase A encoding nucleic acid, and the bovine growth hormone polyadenylation sequence (BGH pA).

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

Example 2

(45) Transient Expression and Analytical Characterization

(46) E. Coli:

(47) 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.

(48) 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.

(49) HEK:

(50) 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.).

(51) 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.

(52) 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

(53) Sortase Mediated Transamidation in Deep Eutectic Solvents

(54) DES-1 in this example is choline chloride mixed with glycerol at a molar ratio of 1:2.

(55) As substrates the following components were used: sortase-motif containing compound: LCR640-ULPETGGRRC (SEQ ID NO: 175) nucleophile: GGGWW-BHQ2 (SEQ ID NO: 46)

(56) The two substrates which have a low solubility in water but good in DES were incubated with two different sortases and the activity determined. Sortase 1: soluble Staphylococcus aureus Sortase A (SEQ ID NO: 05) Sortase 2: soluble Listeria monocytogenes Sortase A (SEQ ID NO: 38)

(57) The educts were dissolved in DES-1 (glycerol and choline chloride 2:1) to a final concentration of 0.5 mM. 1 mM Sortase 1 was stored in 50 mM Tris*HCl pH 7.5, 150 mM NaCl and 10 mM CaCl. This two solutions where mixed 19:1 (v/v) and incubated for 18 hours at 37° C.

(58) Reaction mixture (10 μl) was 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. 1.

(59) The Analysis of the reaction mixture with LC-MS shows in peak 4 the product of the sortase reaction.

Example 4

(60) Sortase Mediated Transamidation in Aqueous Solution

(61) A reaction mixture comprising 0.5 mM of the polypeptide LCR640-ULPETGGGRRC (LCR640 fluorophore conjugated to beta alanine (U); SEQ ID NO: 45) 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 h hours.

(62) The reaction was analyzed without stopping.

(63) 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.

(64) 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.

(65) The respective fragment pattern and masses are shown in the following Table. Table discloses SEQ ID NOS 45, 4, 179, 4, 4, and 180, respectively, in order of appearance.

(66) TABLE-US-00013 LCR-----U-------L------P-----E----T-------G----G----G----R-----R---C-NH 1840 726 1114 1237 (1254) 603 726 512 603 1 Educt: 1840 LPETG 2 LCR- 1329 GGRR 3 Educt: 3676 LPETG dimer 4 Educt: 830 G-Bio 5 Educt: 919 C-Bio 6 LCR- 2067 LPETG-Bio 7 LCR- 2156 LPETC-Bio

Example 5

(67) Kinetic Assays

(68) Two deep eutectic solvents have been tested in this example. DES-1 is choline chloride mixed with glycerol at a molar ratio of 1:2. DES-2 is choline chloride mixed with ethylene glycol at a molar ratio of 1:3.

(69) The mixture comprising the 2 compounds was heated slowly over a flame and shaken until a clear, uniform solution was formed.

(70) The liquid was then allowed to cool to room temperature. The yield was quantitative and the product had a melting point lower than room temperature.

(71) Reaction 1:

(72) TABLE-US-00014 Ratio Components 1 1 mM St. au. SrtA (SEQ ID NO: 05), 50 mM Tris*HCl, pH 7.5, 200 mM NaCl, 10 mM CaCl.sub.2 in water 33 60 μM C-terminally biotinylated oligoglycine (GGGG, SEQ ID NO: 30) in DES 16.5 60 μM glucose dehydrogenase (containing one of the substrates of the sortase reaction (LPXTG; SEQ ID NO: 04) in 50 mM Tris*HCl, pH 7.5, 200 mM NaCl, 10 mM CaCl.sub.2 in water. 49.5 DES
Reaction 2:

(73) TABLE-US-00015 Ratio Components 10 1 mM Listeria monocytogenes SrtA (SEQ ID NO: 38), 50 mM Tris*HCl pH 7.5, 200 mM NaCl, 10 mM CaCl.sub.2 in water 33 60 μM C-terminally biotinylated oligoglycine (GGGG, SEQ ID NO: 30) in DES 16.5 60 μM glucose dehydrogenase (containing one of the substrates of the sortase reaction (LPXTG; SEQ ID NO: 04)) in 50 mM Tris*HCl, pH 7.5, 200 mM NaCl, 10 mM CaCl.sub.2 in water. 40.5 DES

(74) The reaction mixture was incubated at 37° C. for 1, 2, 3 and up to 4 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. 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 (200 mM, pH 5.8) and 50 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.

(75) In comparison the same reaction has been performed in an aqueous buffer system.

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

Example 6

(77) Reporter Immobilization Assay

(78) Determination of Sortase A enzymatic activity can be done using a reporter immobilization assay (REIA) as reported in European Patent application EP14198535 and as outlined below.

(79) Reaction Mixture:

(80) 20 μM polypeptide in question 100 μM nucleophile (GGGG/AAAA/CAAA (SEQ ID NOS 30, 181, and 3, respectively)) 20 μM glucose dehydrogenase with C-terminal sortase motif (LPXTG (SEQ ID NO: 1)) 250 mM MESNA 0.5 mM TCEP.

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

(82) The reaction mixture is prepared in 50 mM Tris-HCl buffer pH 7.5, 150 mM NaCl, 10 mM CaCl.sub.2.

(83) The reaction mixture is incubated at 37° C. for up to 60 hours. The reaction is 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 is centrifuged for 10 min at 5000×g. The supernatant (50 μL) is 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 is incubated for 30 min at 30° C. with shaking at 200 rpm. Thereafter the magnetic beads are 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 are resuspended in 100 μL citrate buffer and 80 μL thereof is 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) are added. The kinetic of the reporter enzyme is measured over a time period of 5 min at 620 nm.

(84) The generation of a signal is showing that the tested polypeptide has sortase A enzymatic activity.

Example 7

(85) Reaction in DIFFERENT SOLVENTS

(86) Trimethylamine, Acetone, Isopropanol, Acetonitrile, Dimethyl sulfoxide, DES (ChCl:Glycerol 1:2, ChCl:Ethylene glycol 1:3 and ChCl:2,3-Butanediol) and water were used as solvents. Beside Hexane the solvents supplemented with (15% water, 200 mM NaCl, 10 mM CaCl.sub.2 and 50 mM Tris/HCl pH 7.5). 0.1 mM Sa-SrtA, 2 mM ULPETGGRR (SEQ ID NO: 174) and 4 mM GGGG-PEG-Biotin (SEQ ID NO: 30) were used. For Hexane the Sa-SrtA was used lyophilized. The reactions were incubated for 6 h at 37° C. at 800 rpm and stopped with the addition of 0.2 M HCl 1:1 (v:v) to the reaction mix. 20 μl was injected on a Aeris C18 Column of an HPLC-system and separated with a 31 min. linear gradient from 2% to 100% buffer B (buffer A (v/v): 95% water, 5% acetonitrile, 0.1% trifluoroacetic acid (TFA); buffer B (v/v): 5% water, 95% Acetonitrile, 0.1% TFA). Peaks were identified using a LC-MS-ESI-system under the same condition.

Example 8

(87) Influence of WATER CONTENT

(88) Mixtures of (ChCl:Glycerol) DES and water (10%, 20%, 30%, 40% and 50%) were prepared and additives were added to a final concentration of (200 mM NaCl, 10 mM CaCl2 and 50 mM Tris/HCl pH 7.5). 0.05 mM Sa-SrtA, 0.5 mM LCRed640-LPETGGRRC (SEQ ID NO: 177) and 5 mM GGGG-PEG-Biotin (SEQ ID NO: 30) was used. The reaction was stopped after 0.5 h, 1.5 h, 2.5 h, 6 h and 20 h with the addition of 0.2 M HCl 1:1 (v:v) with the reaction mix. 20 μl was injected on a Aeris C18 Column of an HPLC-system and separated with a 31 min. linear gradient from 2% to 100% buffer B (buffer A (v/v): 95% water, 5% acetonitrile, 0.1% trifluoroacetic acid (TFA); buffer B (v/v): 5% water, 95% acetonitrile, 0.1% TFA). Peaks were identified using a LC-MS-ESI-system. Retention times: 8.1 min Sort-tag (ULPETGGRR (SEQ ID NO: 174)), 8.6 min Nucleophile (GGGG-Peg-Biotin (SEQ ID NO: 30)), 10.2 ligation product (ULPETGGGG-Peg-Biotin (SEQ ID NO: 182)) and 13.5 min Sa-SrtA.

Example 9

(89) Coupling of Two Lipophilic Substrates

(90) A buffer containing 25% and 100% water in DES (ChCl:Glycerol) with (200 mM NaCl, 10 mM CaCl2 and 50 mM Tris/HCl pH 7.5) was used and 0.15 mM of both substrates (LCRed640-LPETGGRRC (SEQ ID NO: 177) and GGWWK-BHQ2 (SEQ ID NO: 178)) were weighed in. The mixture was shaken for 1 h at 40° C. and centrifuged. To start the reaction 30 μM Sa-SrtA were added to the supernatant and incubated at 37° C. At indicated time points (20 μl) was injected on a Aeris C18 Column of an HPLC-system and separated with a 8 min. linear gradient from 5% to 48% and in 15 min from 48% to 95% buffer B (buffer A (v/v): 95% water, 5% acetonitrile, 0.1% trifluoroacetic acid (TFA); buffer B (v/v): 5% water, 95%, 0.1% TFA). Peaks were identified using a LC-MS-ESI-system.

(91) 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.