Trypsin variants with improved enzymatic properties

Abstract

The present invention relates to trypsin variants with improved enzymatic properties, in particular to mutant trypsin comprising amino acid substitutions at at least two amino acid positions leading to increased affinity for nucleophilic substrates and / or amino acid substitutions at at least two amino acid positions leading to decreased hydrolytic activity.

Description

Technical Field

[0001] The present invention relates to trypsin variants having improved enzymatic properties. Background Art

[0002] There is a pressing need to provide polypeptides having covalent modifications and methods for introducing specific covalent modifications into polypeptides.

[0003] In addition to various purely chemical methods, which are generally non-regiospecific or result in global modification of the polypeptide, there are also molecular biological and enzymatic methods, or a combination of chemical and enzymatic methods, for site-specific modification of polypeptides.

[0004] Without prior genetic manipulation of the polypeptide, site-specific modification of the corresponding polypeptide can only occur in exceptional cases. For example, a recognition sequence for subsequent enzymatic modification is genetically incorporated into the polypeptide sequence, which can be used for position-specific modification (the position of the introduced tag is determined by the position of the recognition sequence).

[0005] In addition to the single modification of polypeptides in a site-specific manner, the orthogonal double modification of polypeptides using enzymes from only one source represents a novelty that cannot be achieved using current technology. In the context of orthogonal double modification, the term "orthogonal" refers to the modification of a polypeptide at two different recognition sequences using two different biocatalysts from the same source without significant cross-reactivity. Enzymatic methods for modifying polypeptides exploit the inherent properties of enzymes, such as the recognition of certain amino acid sequences or functionalities after introduction of the corresponding recognition sequence by site-directed mutagenesis. Regiospecificity results from the high substrate specificity of the corresponding enzymes. The fact that each polypeptide modified in this way has only one recognition sequence gives the method regio- and chemoselective characteristics, since typically only one amino acid is modified within the consensus sequence.

[0006] Protease can be a useful enzyme for modifying polypeptides. Trypsin is a serine protease that specifically cuts the carboxyl terminus of basic amino acid residues. The active site is composed of Ser195, Asp102 and His57 (catalytic triad). Ser195 forms an acyl enzyme intermediate with the substrate to be cut, and therefore significantly participates in protease reactivity. This acyl enzyme intermediate can be attacked by variable nucleophiles (such as water (peptide hydrolysis), amines (peptide aminolysis), alcohols and thiols (peptide (thio) esterification)). By forming a covalent acyl enzyme intermediate, the serine protease trypsin therefore meets all requirements of kinetically controlled acyl transfer.

[0007] In contrast to peptide cleavage, peptide ligation is a two-substrate reaction. The acyl donor binds at the S-binding site of the enzyme, while the acyl acceptor interacts with the S' binding region.

[0008] The C-terminal modification of a polypeptide via a stable amide bond is based on transamidation. The C-terminal region of the labeled polypeptide forms an acyl-enzyme intermediate with a trypsin variant, which can then be nucleophilically attacked by a labeled acyl acceptor.

[0009] In WO 2006 / 015879 A1, the trypsin variant K60E / D189K / N143H / E151H (Trypsiligase I) is described, which recognizes the histidine side chain induced by zinc ions at the P2'-position of peptides and, in the presence of zinc ions, specifically hydrolyzes the recognition sequence -YRH - between the amino acids tyrosine and arginine at the restriction site -Tyr-Arg-His.

[0010] In EP 18 205 212, trypsin variants are described, which comprise amino acid replacements at positions K60 and D189, and at least one amino acid replacement at position Y39 or Y59. Described preferred trypsin variants are Y39H / Y59H / K60E / D189K (Trypsiligase II). EP 18 205 212 further relates to the purposes of a polypeptide comprising a target polypeptide and a restriction site peptide comprising recognition site Tyr-Arg-Xaa-His, wherein Xaa is any amino acid, wherein the restriction site peptide overlaps with the target polypeptide by the amino acid Tyr of the C-terminal of the target polypeptide as a substrate of a mutant trypsin, as described in EP 18 205 212. Also provided are methods for preparing a C-terminal transamidation target polypeptide and methods for preparing an N-terminal transacylation target polypeptide.

[0011] In view of these trypsin variants, there is a need to provide variants with improved synthetic properties that favor peptide aminolysis over hydrolysis during the deacylation step of the transamidation reaction and are independent of metal ions. Summary of the Invention

[0012] The inventors of the present invention have discovered trypsin variants comprising amino acid substitutions at at least two amino acid positions that result in increased affinity for nucleophilic substrates and / or amino acid substitutions at at least two amino acid positions that result in decreased hydrolytic activity.

[0013] In a preferred embodiment, the trypsin variant comprises an amino acid substitution at least at two amino acid positions selected from Group 1, preferably further comprising an amino acid substitution at at least one amino acid position selected from Group 2, wherein Group 1 comprises H40, A55, S214, G219, A221 and Group 2 comprises R96, K97, L99, N143, E151, S190, Q192; or an amino acid substitution at at least one amino acid position selected from Group 1, wherein Group 1 comprises H40, A55, S214, G219, A221 and Group 2 comprises R96, K97, L99, N143, E151, S190, Q192; or

[0014] an amino acid substitution at least at one amino acid position selected from Group 1 comprising H40, A55, S214, G219, A221, and an amino acid substitution at at least two amino acid positions selected from Group 2 comprising R96, K97, L99, N143, E151, S190, Q192; or

[0015] an amino acid substitution at at least two amino acid positions selected from Group 1 comprising H40, A55, S214, G219, A221, and an amino acid substitution at at least two amino acid positions selected from Group 2 comprising R96, K97, L99, N143, E151, S190, Q192; or

[0016] an amino acid substitution at at least three amino acid positions selected from Group 1 comprising H40, A55, S214, G219, A221, and an amino acid substitution at at least one amino acid position selected from Group 2 comprising R96, K97, L99, N143, E151, S190, Q192; or

[0017] amino acid substitutions at at least three amino acid positions selected from Group 1 comprising H40, A55, S214, G219, A221, and amino acid substitutions at at least two amino acid positions selected from Group 2 comprising R96, K97, L99, N143, E151, S190, Q192; or

[0018] Amino acid substitutions at least at three amino acid positions selected from Group 1, and amino acid substitutions at at least three amino acid positions selected from Group 2, wherein Group 1 comprises H40, A55, S214, G219, A221 and Group 2 comprises R96, K97, L99, N143, E151, S190, Q192.

[0019] The present invention also relates to the use of two different trypsin enzymes for orthogonal double modification of two different recognition sequences, and a method for orthogonal double modification of a substrate using two different trypsin enzyme variants.

[0020] In a preferred embodiment, the use of two different trypsins for orthogonal double modification of two different recognition sequences uses trypsin variant A2C8 as the first enzyme and trypsin variant K7F11 as the second enzyme, or the first enzyme is trypsin variant A2C8 and the second enzyme is trypsin variant K7F11_H39Y / H59Y / K189D, or the first enzyme is Trypsiligase II and the second enzyme is trypsin variant A2C8, or the first enzyme is Trypsiligase II and the second enzyme is trypsin variant K7F11, or the first enzyme is trypsin variant K7F11_H39Y / H59Y / K189D and the second enzyme is trypsin variant A2C8, or the first enzyme is trypsiligase II and the second enzyme is trypsin variant K7F11.

[0021] In a preferred embodiment, the method for orthogonal double modification of a substrate comprises the following steps: a) providing an orthogonal double modified substrate, b) modifying the substrate using a first trypsin variant that recognizes a first recognition sequence, c) modifying the substrate using a second trypsin variant that recognizes a second recognition sequence. Preferably, the first trypsin variant or the second trypsin variant is selected from the group consisting of Trypsiligase II, trypsin variant A2C8, trypsin variant K7F11, and trypsin variant K7F11_H39Y / H59Y / K189D. DETAILED DESCRIPTION

[0022] To facilitate understanding of the present invention, a brief discussion of the terminology used in conjunction with the present invention will be provided. This disclosure uses the terminology of Schechter, J., and Berger, A., Biochem. Biophys. Res. Commun. [Biochemistry and Biophysics Research Communications] 27 (1967) 157-162 to describe the positions of various amino acid residues on peptide substrates and individual binding sites within the active site of the corresponding proteolytic enzyme.

[0023] According to the terminology proposed by Schechter, J. and Berger, A. above, the amino acid residues of the peptide substrate are represented by the letter "P". The amino acid on the N-terminal side of the peptide bond to be cleaved ("cleavage site" or "recognition site") of the substrate is designated as P n ...P3, P2, P1, where P nThe amino acid residues at the C-terminal side of the cleavage site of the peptide substrate are named P1`, P2`, P3`, ... P n `, where P n ` is the amino acid residue farthest from the cleavage site. Therefore, the bond to be cleaved (the "cleavage site" or "recognition site") is the P1-P1` bond.

[0024] The amino acid formula of the substrate of endopeptidases (such as trypsin) is as follows:

[0025] P n -P3-P2-P1-P1`-P2`-P3`P n `

[0026] The nomenclature of the substrate binding sites of endopeptidases is similar to the nomenclature of the amino acid residues of peptide substrates. However, the binding subsite of an endopeptidase is indicated by the letter "S" and can include more than one amino acid residue. The substrate binding site for the amino acid at the N-terminal position of the cleavage site is labeled S. n ..., S3, S2, S1. The substrate binding subsites of the amino acids on the carboxyl side of the cleavage site are named S1', S2', S3', ...S n Thus, in endopeptidases, the S1` subsite interacts with the P1` group of the peptide substrate and the incoming nucleophile.

[0027] The general formula describing the substrate binding site of an endopeptidase is:

[0028] S n -S3-S2-S1-S1`-S2`-S3`S n `

[0029] The S1 binding site binds to the side chain of the penultimate amino acid P1 of the peptide substrate (in the case of the trypsin variant according to the present invention, the amino acid Tyr). The S1′ binding site interacts with the side chain of P1′ (in the case of the present invention, Arg). Similarly, the S2′ binding site interacts with the side chain of the Xaa residue at position P2′.

[0030] The term "variant" refers to a polypeptide having an amino acid sequence that is different to some extent from a native polypeptide sequence. Typically, the variant amino acid sequence will have at least about 80% homology with the corresponding parent trypsin sequence, and preferably, it will have at least about 90%, more preferably at least about 95% homology with such corresponding parent trypsin sequence. Amino acid sequence variants have substitutions, deletions, and / or insertions at certain positions within the amino acid sequence of the native amino acid sequence. Preferably, the sequence homology is at least 96% or 97%.

[0031] "Homology" is defined as the percentage of identical residues in amino acid sequence variants after aligning the sequences and introducing gaps (if necessary to maximize the homology percentage). Methods and computer programs for alignment are well known in the art. One such computer program is "Align2," written by Genentech, Inc., which was filed with user documentation on December 10, 1991, in the U.S. Copyright Office, Washington, D.C. 20559.

[0032] Mutated trypsin

[0033] A first aspect of the present invention provides a mutant trypsin comprising an amino acid substitution at at least one amino acid position selected from the group comprising: H40, A55, R96, K97, L99, N143, E151, S190, Q192, S214, G219, A221 corresponding to positions 23, 38, 78, 79, 81, 123, 131, 172, 174, 192, 196 and 198 of the trypsin sequence as shown in SEQ ID NO: 1, respectively, according to the chymotrypsin nomenclature.

[0034] The person skilled in the art is familiar with the so-called chymotrypsin nomenclature, as described in Hartley, BS, and Shotton, DM, The Enzymes, PD Boyer (ed.), Vol. 3, (1971), pp. 323-373, and has no problem aligning the positions of the variant trypsins (given according to the chymotrypsin nomenclature) with the corresponding positions of the trypsin sequence of SEQ ID No: 1.

[0035] In a preferred embodiment, the mutated trypsin comprises additional amino acid substitutions at both positions K60 and D189, and at least one further amino acid substitution at position Y39 or Y59.

[0036] Position 39 according to the chymotrypsin nomenclature corresponds to position 22 of the sequence of the mature anionic rat trypsin II from Rattus norvegicus as shown in SEQ ID NO: 1.

[0037] Position 59 according to the chymotrypsin nomenclature corresponds to position 42 of the sequence of the mature anionic rat trypsin II from Rattus norvegicus as shown in SEQ ID NO: 1.

[0038] Position 60 according to the chymotrypsin nomenclature corresponds to position 43 of the sequence of the mature anionic rat trypsin II from Rattus norvegicus as shown in SEQ ID NO: 1.

[0039] Position 189 according to the chymotrypsin nomenclature corresponds to position 171 of the sequence of the mature anionic rat trypsin II from Rattus norvegicus as shown in SEQ ID NO: 1.

[0040] Since the person skilled in the art is accustomed to referring to positions with reference to the chymotrypsin nomenclature, in the following, references to specific sequence positions (eg position K60 or simply position 60) are based solely on the position according to the chymotrypsin nomenclature.

[0041] In another preferred embodiment, the mutated trypsin comprises additional amino acid substitutions at both positions K60 and D189, and at least one further amino acid substitution by histidine at position N143 or position E151, which positions, according to the chymotrypsin nomenclature, correspond to positions 43, 171, 123 and 131 of the sequence as shown in SEQ ID NO: 1, respectively.

[0042] Position 60 according to the chymotrypsin nomenclature corresponds to position 43 of the sequence of the mature anionic rat trypsin II from Rattus norvegicus as shown in SEQ ID NO: 1.

[0043] Position 143 according to the chymotrypsin nomenclature corresponds to position 123 of the sequence of the mature anionic rat trypsin II from Rattus norvegicus as shown in SEQ ID NO: 1.

[0044] Position 151 according to the chymotrypsin nomenclature corresponds to position 131 of the sequence of the mature anionic rat trypsin II from Rattus norvegicus as shown in SEQ ID NO: 1.

[0045] Position 189 according to the chymotrypsin nomenclature corresponds to position 171 of the sequence of the mature anionic rat trypsin II from Rattus norvegicus as shown in SEQ ID NO: 1.

[0046] In order to identify relevant mutation sites in trypsin and provide variants with improved properties, the inventors started from two independent enzyme libraries based on the trypsin variant K60E / N143H / E151H / D189K (Trypsiligase I).

[0047] To generate library A, amino acid positions H40, A55, K97, L99, S190, and Q192 were randomized, and to generate library B, positions D95, R96, L99, S214, G219, and A221 were randomized. Selection by phage display and subsequent ELISA-based screening yielded Trypsiligase I variant 2G10 (for library A) and variant 1C11 (for library B) as the best variants in terms of synthetic potential.

[0048] Enzyme kinetic analysis of these two variants (see Table 1 and Figure 1 ) show that the optimization of the synthesis potential has different reasons. Variant 2G10 (Trypsiligase I+H40P, A55S, K97D, L99F, S190S, Q192E) shows a strongly enhanced affinity for nucleophilic RH substrates compared to the original enzyme Trypsiligase I, whereby it preferentially integrates as a nucleophile in competition with water, leading to aminolysis (= formation of the desired product) rather than the undesired hydrolysis.

[0049] In contrast, variant 1C11 (Trypsiligase I + R96V, L99F, S214G, G219S, A221G) exhibited hydrolytic activity reduced to 1 / 20 compared to that of Trypsiligase I, resulting in a strong shift in the relationship between aminolysis and hydrolysis toward aminolysis. This variant failed to demonstrate improved affinity for nucleophiles.

[0050] Attached photos

[0051] Figure 1 : Time course of product formation in transamidation reactions catalyzed by Trypsiligase I and improved variants 2G10, 1C11, and hybrid variants. Reaction conditions: 15 μM Bz-AAYRHAAG-OH (acyl donor), 30 μM H-RHAK-OH (acyl acceptor), 0.5-2.1 μM trypsin variants, 100 mM HEPES / NaOH pH 7.8, 0.1 mM ZnCl2, 100 mM NaCl, 10 mM CaCl2, T = 30°C. UPLC analysis: Waters Acquity Ultra Performance LC, C18 column, gradient 5-40% acetonitrile over 4 minutes, detection at 254 nm.

[0052] Figure 2: Substrate specificity data for selected variants identified during phage display selection and screening of a Trypsiligase II library. 100 μM acyl donor (Bz-PGGXaaXaaXaaXaaAG-OH); 200 μM acyl acceptor (H-XaaXaaXaaAK(DNP)-OH); 1-5 μM enzyme variant; 100 mM HEPES pH 7.8, 100 mM NaCl, 10 mM CaCl2, 0.1 mM ZnCl2, T = 30°C. UPLC analysis: Waters Acquity Ultra Performance LC, C18 column, gradient 5-60% acetonitrile over 5 minutes, detection at 360 nm; (k_(cat,AL)^app): apparent turnover rate of the aminolysis reaction. The amino acid changes of the acyl donor / acyl acceptor pair at the Xaa-position are represented on the y-axis, for example YRAH involves the acyl donor Bz-PGGYRAHAG-OH and the corresponding acyl acceptor H-RAHAK(DNP)-OH.

[0053] Figure 3 : Time course of product formation in transamidation reactions catalyzed by variants A2C8, A2C8_H39Y, A2C8_H59Y, and A2C8_H39Y / H59Y. Reaction conditions: 100 μM Bz PGGYRKKAG-OH (acyl donor), 200 μM H-RKKAK-OH (acyl acceptor), 1 μM trypsin variant, 100 mM HEPES / NaOH pH 7.8, 100 mM NaCl, 10 mM CaCl2, T = 30°C. UPLC analysis: Waters Acquity Ultra Performance LC, C18 column, gradient 5-40% acetonitrile over 4 minutes, detection at 254 nm.

[0054] Figure 4: Substrate specificity data for selected variants identified during phage display selection and screening of a Trypsiligase II library. 100 μM acyl donor (Bz-PGGXaaXaaXaaXaaAG-OH); 200 μM acyl acceptor (H-XaaXaaXaaAK(DNP)-OH); 1-5 μM enzyme variant; 100 mM HEPES pH 7.8, 100 mM NaCl, 10 mM CaCl2, 0.1 mM ZnCl2, T = 30°C; UPLC analysis: Waters Acquity Ultra Performance LC, C18 column, gradient 5-60% acetonitrile over 5 minutes, detection at 360 nm; (k_(cat,AL)^app): apparent turnover rate of the aminolysis reaction. The amino acid changes of the acyl donor / acyl acceptor pair at the Xaa-position are represented on the y-axis, for example YRAH involves the acyl donor Bz-PGGYRAHAG-OH and the corresponding acyl acceptor H-RAHAK(DNP)-OH.

[0055] Figure 5 : Time course of product formation in transamidation reactions catalyzed by variant A2C8_H39Y / H59Y / E60K / K189D and wild-type trypsin with various peptide substrates. Reaction conditions: 100 μM acyl donor (BzPGGXaaXaaXaaHAG-OH), 200 μM acyl acceptor (H-XaaXaaXaaAK(DNP)-OH), 10 μM trypsin variant A2C8_H39Y / H59Y / E60K / K189D or 2.5 nM anionic rat trypsin II, 100 mM HEPES / NaOH pH 7.8, 0.1 mM ZnCl2, 100 mM NaCl, 10 mM CaCl2, T = 30°C. UPLC analysis: Waters Acquity UltraPerformance LC, C18 column, gradient 5-60% acetonitrile over 5 minutes, detection at 360 nm.

[0056] Figure 6:By utilizing the variation of the substrate specificity of two kinds of Trypsiligase II variants, Fab fragment is carried out double modification.Use the HER2 specific Fab fragment (anti-Her2-Fab-LC_RRKH / HC_YRAH, heavy chain (SEQ ID NO:2) and anti-Her2-Fab-LC_RRKH / HC_YRAH, light chain (SEQ ID NO:3)) of trastuzumab (Trastuzumab), and introduce each recognition sequence genetically.A) in the first step, the Trypsiligase II variant K7F11_H39Y / H59Y / K189D using the recognition sequence RRKH is connected with the nucleophile of carboxyfluorescein (CF).B) in the second step, the Trypsiligase II variant A2C8 of the recognition sequence YRAH is used for being modified with the nucleophile being covalently bound to maytansine (DM1).LC-MS analysis.The amount of the modified and unmodified Fab fragment species for the first and second modification steps is shown in M ​​spectrum A and B respectively. MS spectrum A) Peak 1: anti-Her2-Fab-LC_R-OH / HC_YRAH (M 计算 =50116Da, M 发现 =50118Da), Peak 2: Anti-Her2-Fab-LC_RRKH / HC_YRAH (M 计算 =50666Da, M 发现 =50666Da), Peak 3: Anti-Her2-Fab-LC_RRKHAK(CF) / HC_YRAH(M 计算 =51095Da, M 发现 =51097Da). MS spectrum B) Peak 1: Anti-Her2-Fab-LC_RRKHAK(CF) / HC_Y-OH(M 计算 =49417Da, M 发现 =49417Da), Peak 2: Anti-Her2-Fab-LC_R-OH / HC_YRKKAK (MCC-DM1) (M 计算 =50006Da, M 发现 =50000Da), Peak 3: Anti-Her2-Fab-LC_RRKH / HC_YRKKAK (MCC-DM1) (M 计算 =50555Da, M 发现 =50556Da), peak 4: anti-Her2-Fab-LC_RRKHAK(CF) / HC_YRKKAK(MCC-DM1)(M 计算 =50985Da, M 发现=50987Da), Peak 5: Anti-Her2-Fab-LC_RRKHAK(CF) / HC_YRAH(M 计算 =51095Da, M 发现 =51095 Da). First step reaction conditions: 100 μM Fab; 2000 μM RKHAK(CF)-OH; 5 μM K7F11_H39Y / H59Y / K189D; 100 mM HEPES / NaOH pH 7.8, 100 mM NaCl, 10 mM CaCl2, T = 30°C, t = 180 min. Second step reaction conditions: 50 μM Fab; 1000 μM RKKAK(MCC-DM1)-OH; 5 μM A2C8; 100 mM HEPES / NaOH pH 7.8, 100 mM NaCl, 10 mM CaCl2, T = 30°C, t = 40 min.

[0057] Methods and Materials

[0058] UPLC analysis

[0059] Peptide and reaction analysis was performed using a Waters ACQUITY UPLC system equipped with an RP-C18 column (ACQUITY UPLC BEH 130, C18, 1.7 μm, 2.1 × 100 mm) at a flow rate of 0.5 ml / min. The mobile phases used were water with 0.05% TFA (A) and acetonitrile with 0.05% TFA (B), respectively. For analysis, two methods were used:

[0060] Method I: Linear gradient 5% to 40% B in 5 minutes, detection at 254 nm.

[0061] Method II: Linear gradient 5%-60% B in 5 minutes, detection at 360 nm.

[0062] The amounts of product and educt were calculated from the integrated peak areas.

[0063] Mass spectrometry analysis

[0064] Mass spectrometry (MS) analysis was performed by LC-MS using a LC-MS coupled to a Waters ZQ TM Waters HPLC system with MS detector. RP-C8 column (XBridge TMLC separation was performed using a flow rate of 0.3 ml / min using a HPLC-MS / MS setup (C8, 3.5 μM, 2.1 x 100 mm). The mobile phases used were water containing 0.1% TFA (A) and acetonitrile containing 0.1% TFA (B). A linear gradient from 5% to 95% B over 10 minutes was applied for separation, with detection at 220 nm.

[0065] Expression and purification of trypsin variants

[0066] To recombinantly produce all described trypsin variants, the corresponding genes were subcloned into the pPICZαA expression vector using the Agel / XhoI restriction enzyme sites. Escherichia coli DH5α was transformed with the gene encoding vector, and the cells were then plated on LB low salt (5 g / l yeast extract; 10 g / l tryptone; 5 g / l NaCl) agar plates containing 25 μg / ml Zeocin. After incubation at 37°C overnight, single colonies were picked and transferred to LB low salt liquid medium containing 25 μg / ml Zeocin. The cells were incubated at 37°C overnight with continuous shaking. The cells were collected by centrifugation at 5000xg for 5 minutes, and the plasmids were then isolated according to standard experimental procedures. The isolated plasmids were linearized by SacI digestion. The linearized plasmid was then transformed into Pichia pastoris X-33 cells by electroporation and plated on YPDS (10 g / l yeast extract; 20 g / l peptone; 20 g / l dextrose; 1 M sorbitol) agar plates containing 100 μg / ml Zeocin. The plates were incubated at 30°C for three days. To express the trypsin variants, individual colonies were picked and transferred to buffered minimal medium (100 mM potassium phosphate pH 6.0; 1.34% yeast nitrogen base) containing 2% dextrose. After incubation at 30°C and continuous shaking for 48 hours, the cells were harvested at 4000 x g for 5 minutes. Afterwards, the cell pellet was resuspended in buffered minimal medium and protein production was induced by adding 1% (v / v) methanol, while the trypsin variant was secreted into the supernatant. Protein production was performed by adding 1% (v / v) methanol every day and incubating at 30°C for 5 days under continuous shaking. After five days, the cells were separated from the supernatant by centrifugation at 5000 x g for 20 minutes. To isolate the secreted trypsin variant, a two-step purification was performed consisting of cation exchange chromatography followed by size exclusion chromatography. Using an AKTA FPLC, 20 ml of HiPrep TMA SP FF column (GE Healthcare) was equilibrated with 10 column volumes of binding buffer (20 mM sodium acetate pH 4.0). The supernatant was diluted with 1 volume of binding buffer and loaded onto the column. After a wash step using 10 column volumes of binding buffer, the protein was eluted with elution buffer (100 mM HEPES / NaOH pH 7.8, 200 mM NaCl, 10 mM CaCl2) and the protein-containing fractions were detected by absorption at 280 nm. The combined protein-containing fractions were concentrated to a volume of approximately 1 ml using a centrifugal filter device (NMWL: 10 kDa, Millipore). The concentrated protein solution was then purified by size exclusion chromatography using a HiLoad elution buffer (100 mM HEPES / NaOH pH 7.8, 100 mM NaCl, 10 mM CaCl). TM 16 / 60Superdex TM Purification was performed using a 75 pg column (GE Healthcare). Protein-containing fractions were identified by SDS-PAGE as being associated with a monomeric enzyme species (approximately 24 kDa) with an absorption peak at 280 nm. Fractions with a purity > 90% were pooled and purified using The protein was concentrated using a centrifugal filter device (NMWL: 10 kDa, Millipore). Protein concentration was determined by absorbance of the variants at 280 nm and the corresponding extinction coefficient. The identity of the trypsin variants was confirmed by LC-MS.

[0067] Expression and purification of Fab fragments

[0068] In order to recombinantly produce the Her2-specific Fab fragment anti-Her2-Fab-LC_RRKH / HC_YRAH, the corresponding gene sequence (Seq. ID No. 64) was subcloned into the pASK-IBA7Plus expression vector by standard methods. Escherichia coli BL21 (DE3) was transformed with the expression plasmid and plated on LB (5 g / l yeast extract; 10 g / l tryptone; 10 g / l NaCl) agar plates containing 100 μg / ml ampicillin. After incubation at 37°C overnight, single colonies were picked and transferred to LB liquid medium containing 100 μg / ml ampicillin. To express the Fab fragment, the preculture was incubated at 37°C with continuous shaking overnight and then used in LB medium containing 100 μg / ml ampicillin (starting OD 600nm When the culture reaches OD 600nmWhen the pH value was 0.8-1, protein production was induced by adding 0.2 μg / ml anhydrotetracycline. After incubation at 30°C for 4 hours under continuous shaking, the cells were harvested by centrifugation at 5000xg for 20 minutes. The pellet was resuspended in lysis buffer (20mM sodium phosphate pH 7.0, 0.1mM AEBSF). The cells were disrupted by ultrasonic treatment (8×10 seconds, 30% amplitude) and cell debris was removed by ultracentrifugation at 20000xg for 35 minutes. In order to isolate the Fab fragment, a two-step purification consisting of protein G affinity chromatography followed by size exclusion chromatography was performed. Using AKTA FPLC, 1ml HiTrap TM A Protein GHP column (General Health Medical Group) was equilibrated with 10 column volumes of binding buffer (20 mM sodium phosphate pH 7.0). After loading the supernatant onto the column, a wash step was performed with 10 column volumes of binding buffer. Elution was performed with 10 column volumes of elution buffer (100 glycine / HCl pH 2.7), and the collected fractions were immediately neutralized with 20% (v / v) neutralization buffer (1 M Tris pH 9.0). Protein-containing fractions were detected by absorbance at 280 nm. The combined protein-containing fractions were concentrated to a volume of approximately 1 ml using a centrifugal filter device (NMWL: 10 kDa, Millipore). The concentrated protein solution was then purified by size exclusion chromatography using a HiLoad elution buffer (100 mM HEPES / NaOH pH 7.8, 100 mM NaCl, 10 mM CaCl2). TM 16 / 60Superdex TM Purification was performed on a 75 pg column (GE Healthcare). Fractions associated with the absorption peak at 280 nm of the monomeric Fab species (approximately 50 kDa) were identified by SDS-PAGE. Fractions with a purity > 90% were pooled and purified using The protein was concentrated using a centrifugal filter device (NMWL: 10 kDa, Millipore). The final protein concentration was determined by absorbance at 280 nm and the corresponding extinction coefficient. The identity of the Fab fragment was confirmed by LC-MS.

[0069] Peptide synthesis

[0070] All reagents and detergents used for peptide synthesis were purchased from Sigma All amino acids and building blocks (Lys(Dnp), Lys(ivDDE)) used for peptide synthesis were purchased from Marktredwitz, Germany. Biotech GmbH. DM1 was purchased from San Diego, USA

[0071] The peptide was synthesized by standard procedures using the Fmoc / protecting group strategy as described by Merrifield. The first amino acid was coupled to a chlorotrityl resin according to the final peptide sequence. Next, Fmoc cleavage was performed using 20% ​​piperidine in DMF. For further coupling, the amino acids were activated using ((1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate) (HATU). Final release from the resin and deprotection of the side chain protecting groups were achieved by using 95% TFA, 2.5% triisopropylsilane and 2.5% water. After evaporation of the solvent, the oily residue was dissolved in water / ACN and purified by preparative HPLC (Merck / Hitachi-HPLC, Vydac-C18, 5-80% ACN, 30 / 60 min). After freeze-drying, the product containing the peptide fraction was obtained as a crystalline powder. The identity and purity of the products were confirmed by HPLC (Waters, ACQUITY UPLC, BEH130) and LC-MS (Waters, X-Bridge BEH300). The purity of all peptides was higher than 98%.

[0072] To synthesize the maytansine (DM1) functionalized peptide H-RKKAK (MCC-DM1) -OH, the building block Fmoc-Lys (ivDde) ​​was used. After the synthesis of the RKKAK (ivDde) ​​peptide, the lysine protecting group was removed from the fully protected peptide using 2% hydrazine in DMF. The lysine side chain was then functionalized with 1.1 equivalents of succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate in DMF. In the final step, RKKAK (MCC) was coupled to maytansine via a Michael addition reaction using a buffered phosphate buffer / ACN system (pH 7.4).

[0073] Final release from the resin, deprotection of the side chain protecting groups, and purification of H-RKKAK(MCC-DM1)-H were completed as described previously. The product was obtained as a mixture of two isomers. Product identity and purity were verified by UPLC and LC-MS. The purity of H-RKKAK(MCC-DM1)-OH was greater than 99%.

[0074] Model transamidation reactions catalyzed by Trypsiliqase II and improved Trypsiliqase II variants

[0075] Model transamidation reactions were performed at 30°C in a solution containing 15 to 250 μM acyl donor, 2 equivalents of the corresponding acyl acceptor, varying concentrations of trypsin variants, 100 mM HEPES / NaOH pH 7.8, ±0.1 mM ZnCl2, 100 mM NaCl, 10 mM CaCl2. The acyl donor and acyl acceptor had identical amino acids at the P1'-P3' positions, e.g., Bz-PGGY RAH AG+H- RAH AK-OH or Bz-PGGY RKK AG+H- RKK AK-OH. Variations are indicated. The acyl acceptor may carry a 2,4-dinitrophenyl (DNP) group at the side chain of the terminal lysine, e.g., H- RAH AK(DNP)-OH or H-RKKAK(DNP)-OH. The reaction was initiated by adding the enzyme. To record the time course of product formation and / or for kinetic evaluation of the reaction, several aliquots of the reaction mixture were quenched with 25% (v / v) acetic acid at various time points over a period of up to 4 hours. The composition of the reaction mixture was analyzed by UPCL using Method I or II, depending on the absence (Method I) or presence (Method II) of 2,4-dinitrophenyl in the acyl acceptor (see Example 1). The measurements were repeated with an error of less than 5%.

[0076] Determining the turnover of hydrolysis reactions catalyzed by Trypsiligase II and improved Trypsiliqase II variants Rate

[0077] The hydrolysis reaction was carried out at 30°C in a solution containing varying concentrations of the acyl donor Bz-PGGYRAHAG-OH (0-5000 μM for Trypsiligase II and 0-1500 μM for the A2C8 variant), 2 μM trypsin variant, 100 mM HEPES / NaOH pH 7.8, 100 mM NaCl, 10 mM CaCl2. In the case of Trypsiligase II, the reaction was carried out in the presence of 100 μM ZnCl2. The reaction was initiated by the addition of the enzyme. For kinetic evaluation of the reaction, several aliquots of the reaction mixture were quenched with 25% (v / v) acetic acid at different time points over 30 minutes. The composition of the reaction mixture was analyzed by UPLC using Method I (see Example 1). The UV absorption of the N-terminal benzoyl group (Bz-) present in the acyl donor and the hydrolysis product was detected at 254 nm. In order to determine the turnover rate (k cat,HL ), the initial rate of the hydrolysis reaction was plotted against the corresponding acyl donor concentration and fitted to the Michaelis-Menten equation. The measurements were repeated with an error of less than 5%.

[0078] K of nucleophilic peptide M Determination of value

[0079] For Trypsiligase II, due to its strong dependence on zinc ions, the K of the nucleophilic peptide (acyl acceptor) is M The values ​​were measured in the presence or absence of zinc ions. In the case of variant A2C8, the measurements were performed in the absence of zinc ions.

[0080] The transamidation reaction using Trypsiligase II and variant A2C8 was performed at 30°C in a solution containing 250 μM acyl donor Bz-PGGYRAHAG-OH, varying concentrations of acyl acceptor H-RAHAK(DNP)-OH (0-5000 μM for Trypsiligase II in the absence of zinc ions, 0-1500 μM for Trypsiligase II in the presence of zinc ions, and 0-1000 μM for A2C8 variants), 0.2-1.5 μM trypsin variants, 100 mM HEPES / NaOH pH 7.8, ±0.1 mM ZnCl2, 100 mM NaCl, 10 mM CaCl2. The reaction was initiated by adding the enzyme. For kinetic evaluation of the reaction, several aliquots of the reaction mixture were quenched with 25% (v / v) acetic acid at different time points over 30 minutes. The composition of the reaction mixture was analyzed by UPLC using Method II (see Example 1). The UV absorption of 2,4-dinitrophenyl (DNP) present in the acyl acceptor and the aminolysis product was detected at 360 nm. M The initial rate of the aminolysis reaction was plotted against the corresponding acyl acceptor concentration and fitted to the Michaelis-Menten equation. The measurements were repeated with an error of less than 5%.

[0081] Orthogonal dual modification of Her2-specific Fab fragments

[0082] In order to double modify the Her2-specific Fab fragment, two orthogonal recognition sequences are attached to the corresponding C-termini of the heavy chain and light chain, respectively. This is done to achieve enzyme-mediated coupling of two different functional groups. As shown in Seq. ID No. 2, the light chain is extended at the C-terminus by a short peptide spacer (LSPGG), followed by the amino acid sequence RRKHAG, which contains the recognition sequence (RRKH) of the variant K7F11_H39Y / H59Y / K189D. The C-terminus of the heavy chain is extended by a short peptide spacer (ADKPGG), followed by the amino acid sequence YRAHAG, which contains the recognition sequence (YRAH) of the variant A2C8 and a cMyc-tag (EQKLISEEDL) for optional purification or detection purposes. Double modification of a Her2-specific Fab fragment containing two orthogonal recognition sequences (aHer2-Fab-LC_RRKH-HC_YRAH) was performed by a two-step modification reaction, in which a fluorescent dye (5(6)-carboxyfluorescein) was attached to the light chain in the first step and a cytotoxic compound maytansine (DM1) was attached to the heavy chain in the second step. Figure 6 ).

[0083] In the solution comprising 100 μM aHer2-Fab-LC_RRKH / HC_YRAH, 2000 μM H-RKHAK (CF) -OH, 5 μM K7F11_H39Y / H59Y / K189D, 100 mM HEPES / NaOH pH 7.8, 100 mM NaCl, a transamidation reaction for modifying the light chain was carried out. The reaction was initiated by adding enzyme and incubated at 30°C for 180 minutes. Subsequently, the enzyme and the remaining nucleophile (H-RKHAK (CF) -OH) with carboxyfluorescein were removed by protein G affinity chromatography. Using AKTA FPLC, 1 ml HiTrap TM A Protein G HP column (GE Healthcare) was equilibrated with 10 column volumes of binding buffer (20 mM sodium phosphate pH 7.0). After application of the reaction mixture, the column was washed with 10 column volumes of binding buffer. The bound Fab fragment species were then eluted with 10 column volumes of elution buffer (100 glycine / HCl pH 2.7), and the collected fractions were immediately neutralized with 1 / 5 volume of neutralization buffer (1 M Tris pH 9.0). The protein-containing fractions were pooled and eluted using The buffer was exchanged into 100 mM HEPES / NaOH pH 7.8, 100 mM NaCl using a centrifugal filter device (NMWL: 10 kDa, Millipore).

[0084] The second modification reaction of the heavy chain was carried out in a solution containing 50 μM single modified aHer2-Fab-LC_RRKHAK(CF) / HC_YRAH, 1000 μM H-RKKAK(MCC-DM1)-OH, 5 μM A2C8, 100 mM HEPES / NaOH pH 7.8, 100 mM NaCl. The reaction was initiated by adding the enzyme and incubated at 30°C for 40 minutes. Subsequently, the enzyme and the remaining nucleophile with DM1 (H-RKHAK(MCC-DMI)-OH) were removed by protein G affinity chromatography as described above. The ratio of the modified and unmodified Fab fragment species of the first and second modification steps was analyzed by LC-MS (see Example 1). After the first modification step, up to 90% of the Fab fragments were exclusively modified with carboxyfluorescein on the light chain (anti-Her2-Fab-LC_RRKHAK(CF) / HC_YRAH, Figure 6 Spectrum A peak 3, M 计算 =51095Da, M 发现 =51097Da). In addition, two minor byproducts could be identified, corresponding to unconsumed Fab fragments (anti-Her2-Fab-LC_RRKH / HC_YRAH, Figure 6 Spectrum A peak 2, M 计算 =50666Da, M 发现 =50666Da) and a Fab fragment with a hydrolysis recognition sequence on the light chain (anti-Her2-Fab-LC_R_OH / HC_YRAH, Figure 6 Spectrum A peak 1, M 计算 =50116Da, M 发现 =50118 Da). No modifications to the recognition sequence on the heavy chain were detected.

[0085] After the second modification step, up to 75% of the Fab fragments were doubly modified, i.e., with carboxyfluorescein on the light chain and DM1 on the heavy chain (anti-Her2-Fab-LC_RRKHAK(CF) / HC_YRKKAK(MCC-DM1), Figure 6 Spectrum B peak 4, M 计算 =50985Da, M 发现 =50987 Da) , which represents the expected configuration. In addition, four minor byproducts were detected, corresponding to single modified Fab fragments with carboxyfluorescein on the light chain and full-length recognition sequence on the heavy chain (anti-Her2-Fab-LC_RRKHAK(CF) / HC_YRAH, Figure 6 Spectrum B peak 5, M 计算 =51095Da, M 发现=51095Da), a single modified Fab fragment with DM1 on the heavy chain and the full-length recognition sequence on the light chain (anti-Her2-Fab-LC_RRKH / HC_YRKKAK (MCC-DM1), Figure 6 Spectrum B peak 3, M 计算 =50555Da, M 发现 =50556Da), a single modified Fab fragment with DM1 on the heavy chain and a hydrolysis recognition sequence on the light chain (anti-Her2-Fab-LC_R-OH / HC_YRKKAK (MCC-DM1), Figure 6 Spectrum B peak 2, M 计算 =50006Da, M 发现 =50000 Da) and a single modified Fab fragment with carboxyfluorescein on the light chain and a hydrolysis recognition sequence on the heavy chain (anti-Her2-Fab-LC_RRKHAK(CF) / HC_Y-OH, Figure 6 Spectrum B peak 1, M 计算 =49417Da, M 发现 =49417 Da). Again, no modification was detected at the C-terminus of the light chain, which may be related to variant A2C8.

[0086] Example

[0087] Example 1

[0088] The product yield and apparent turnover rate of aminolysis were calculated by model transamidation reaction under the following conditions: and the apparent turnover rate of hydrolysis Measurement of K of acyl acceptor: 250 μM Bz-AAYRHAAG-OH (acyl donor), 500 μM H-RHAK-OH (acyl acceptor), 5-10 μM trypsin variant, 100 mM HEPES / NaOH pH 7.8, 0.1 mM ZnCl2, 100 mM NaCl, 10 mM CaCl2, T = 30°C. M The values ​​were determined by measuring the apparent turnover rate of aminolysis at a constant acyl donor concentration and a varying acyl acceptor concentration.

[0089] Table 1: Summary of enzymatic parameters of Trypsiligase I and improved variants 2G10 and 1C11.

[0090]

[0091] Compared to native trypsiligase (222 μM), 2G10 showed an increased affinity for acyl acceptors (23 μM), and 1C11 had a reduced affinity for acyl acceptors (>5000 μM). Both improved variants had a significantly increased ratio of aminolysis to hydrolysis activity (factors of 6 and 13 for 2G10 and 1C11 respectively). This is related to the improved synthesis efficiency reflected by the increased product yield. For 2G10, the improved ratio of aminolysis to hydrolysis is the result of having a better affinity for peptide nucleophiles. This is accompanied by direct competition between water molecules and peptides for the nucleophilic attack on the acyl-enzyme-intermediate in the deacylation step of the transamidation reaction. For 1C11, the improved ratio of aminolysis to hydrolysis is the result of a significant reduction in hydrolysis activity (factor of 37), while the aminolysis activity is reduced to 1 / 3. The 2G10 and 1C11 mutations were combined in one variant (hybrid) to test whether there was a synergistic effect on synthetic efficiency due to different improvements.

[0092] Example 2

[0093] Based on these enzyme kinetic observations, the positions identified in the two variants 2G10 and 1C11 have been combined into a hybrid variant. Figure 1 As shown, the hybrid variant resulting from the combination of the positions identified in variants 2G10 and 1C11 benefits from two positive effects.

[0094] The product yield and apparent turnover rate of aminolysis were calculated by model transamidation reaction under the following conditions: and the apparent turnover rate of hydrolysis Measurement of: 15 μM Bz-AAYRHAAG-OH (acyl donor), 30 μM H-RHAK-OH (acyl acceptor), 0.5-2.1 μM trypsin variants, 100 mM HEPES / NaOH pH 7.8, 0.1 mM ZnCl2, 100 mM NaCl, 10 mM CaCl2, T = 30°C.

[0095] Table 2: Summary of enzymatic parameters of Trypsiligase I, 2G10, 1C11 and hybrid variants.

[0096]

[0097] Heterozygous variants show a further improvement in synthesis efficiency, which is reflected in the improvement in product yield, because the ratio of aminolysis to hydrolysis is significantly improved, particularly under low substrate concentrations (15 μM). This concludes that there is a synergistic effect, thereby producing such trypsin variants, which show better synthesis characteristics than natural Trypsiligase I and improved variants 2G10 and 1C11. In order to generate new trypsin variants with changed recognition sequences, based on Trypsiligase II, a new trypsin library was designed, which comprises the amino acid position showing improvement in the I synthesis efficiency of Trypsiligase. The library is selected by phage display to enrich the potentially improved transamidase using two different substrates with recognition sequences YRAH and YRKH.

[0098] Example 3

[0099] Based on these studies, the mutation positions of these two variants, 2G10 and 1C11, have been combined in a new enzyme library based on the trypsin variant Y39H / Y59H / K60E / D189K (Trypsiligase II), so that the variants obtained from this library benefit from two positive effects and are therefore further optimized. Therefore, in this Trypsiligase II library, positions 40, 55, 96, 97, 143, 151, 190, 192, 214, 219 and 221 are randomized, while the mutation L99F is fixed because it is present in both Trypsiligase I libraries A and B. Positions 143 and 151 in Trypsiligase I are responsible for zinc complexation and therefore convey histidine specificity for the recognition sequence YRH. In Trypsiligase II, this histidine specificity is shifted by positions 39 and 59, resulting in the recognition sequence YRAH. Therefore, positions 143 and 151, which are potentially responsible for the specificity of the P2' position, can be used for randomization in the trypsin II library. In addition to the desired effect of increasing the affinity for substrates / nucleophiles, this can also lead to possible independence of zinc complexation, which is desirable for application-oriented modification of recombinant proteins. Using phage display and ELISA-based screening, the trypsiligase II variants shown in Tables 3 and 4 have been identified.

[0100] Table 3: YR in use ASummary of variants identified during phage display selection and screening of a Trypsiligase II library selected for H substrates, along with associated data on enzyme activity and maximum product yield. 100 μM Bz-PGGYR(A / K)HAG-OH; 200 μM R(A / K)HAK-OH; 100 mM HEPES pH 7.8, 100 mM NaCl, 10 mM CaCl2, 100 μM ZnCl2, T = 30°C; 4Tn II = Trypsiligase II; P max =maximum yield (%); A S [AL] = specific aminolytic activity; A S [HL] = specific hydrolytic activity; A S A / H = quotient of aminolysis activity / hydrolysis activity; YRAH / YRKH = quotient of maximum product yield of YRAH substrate / maximum product yield of YRKH substrate.

[0101]

[0102] Table 4: YR-containing K Summary of variants identified during phage display selection and screening of a Trypsiligase II library selected for H substrates, along with associated data on enzyme activity and maximum product yield. 100 μM Bz-PGGYR(A / K)HAG-OH; 200 μM R(A / K)HAK-OH; 100 mM HEPES pH 7.8, 100 mM NaCl, 10 mM CaCl2, 100 μM ZnCl2, T = 30°C; 4Tn II = Trypsiligase II; P max =maximum yield (%); A S [AL] = specific aminolytic activity; A S [HL] = specific hydrolytic activity; A S A / H = quotient of aminolysis activity / hydrolysis activity; YRAH / YRKH = quotient of maximum product yield of YRAH substrate / maximum product yield of YRKH substrate.

[0103]

[0104] In order to identify improved biocatalysts, two variant pools selected in the 4th round by phage display were subjected to high-throughput screening based on ELISA. A total of 26 trypsin variants were identified that showed improved synthesis efficiency when using either YRAH or YRKH substrates. No variants were identified that clearly distinguished between the two recognition sequences.

[0105] Example 4

[0106] The most promising variants identified by phage display selection were further characterized with respect to substrate specificity, e.g. Figure 2 shown.

[0107] Variants A2C8 and K7F11 showed the highest activity against the YRKK substrate sequence. A high degree of flexibility in specificity for the P3' position was observed across all variants. This leads to the assumption that the zinc ion is redundant for the optimized variants. Furthermore, variants A2C8 and K7F11 may be potential orthogonal biocatalysts, as they possess orthogonal pairs of recognition sequences. A2C8 accepts LRKH as an acyl donor, while K7F11 does not. K7F11 accepts WRAH as an acyl donor, while A2C8 does not.

[0108] For the most promising variants, A2C8 (Trypsiligase II + H40F, A55A, R96E, K97D, L99F, N143E, E151Y, S190V, Q192A, S214G, G219Q, A221T) and K7F11 (Trypsiligase II + H40Y, A55A, R96E, K97E, L99F, N143V, E151 E, S190A, Q192V, S214G, G219P, A221Q), further studies were performed on metal ion dependence and synthesis efficiency.

[0109] Table 5 shows the results regarding the zinc dependence of the transamidation reactions catalyzed by Trypsiligase II, A2C8 and K7F11. Table 6 shows the results of kinetic measurements for Trypsiligase II and variants A2C8 and K7F11.

[0110] Table 5: Study of the zinc dependence of transamidation reactions catalyzed by Trypsiligase II, A2C8, and K7F11. Product yields and apparent turnover rates of aminolysis in model transamidation reactions in the presence or absence of zinc ions. and the apparent turnover rate of hydrolysis A2C8 and K7F11 reaction conditions: 100 μM Bz-PGGYRX aa X aa AG-OH (acyl donor), 200 μM H-RX aa X aaAK(DNP)-OH (acyl acceptor), 2 μM trypsin variant, 100 mM HEPES / NaOH pH 7.8, ±0.1 mM ZnCl2, 100 mM NaCl, 10 mM CaCl2, T = 30°C. Trypsiligase II reaction conditions: 250 μM Bz-AAYRAHAG-OH (acyl donor), 500 μM H-RAHAK(DNP)-OH (acyl acceptor), 2 μM trypsin variant, 100 mM HEPES / NaOH pH 7.8, ±0.1 mM ZnCl2, 100 mM NaCl, 10 mM CaCl2, T = 30°C. UPLC analysis: Waters Acquity Ultra Performance LC, C18 column, gradient 5-60% acetonitrile over 5 minutes, detection at 360 nm; R1: Bz-PGG, R2: AG-OH

[0111]

[0112] Natural Trypsiligase II shows a strong dependence on zinc ions. In the absence of zinc ions, the apparent turnover rate of the aminolysis reaction is reduced to 1 / 12, which causes the product yield to drop from 18% to 4%. The reason is that the affinity for nucleophiles is reduced due to the lack of complexation between the artificial histidine and the peptide-located histidine of Trypsiligase II (H39 and H59) with zinc ions as the central atom. When using two substrates with YRAH and YRKH sequences, A2C8 did not show dependence on zinc ions, while the apparent turnover rate of the aminolysis reaction benefited from the absence of zinc ions. This allows the substitution of histidine at the P3' position, as shown for the YRKK substrate, which has an additional effect on the turnover rate and product yield of the aminolysis reaction. K7F11 only shows a slight dependence on zinc ions for the YRAH substrate. In the absence of zinc ions, the turnover rate of the aminolysis reaction is reduced to 1 / 2, which causes the product yield to drop from 43% to 26%. This dependency can be eliminated by substituting lysine for alanine at the P2' position, resulting in improved synthetic performance in the absence of zinc ions. Furthermore, K7F11 tolerates substitution with histidine at the P3' position, which also has an increased impact on the apparent turnover rate of the aminolysis reaction and product yield. The lack of zinc dependency in K7F11 and A2C8 leads to the hypothesis that the artificial histidine in trypsiligase II can be reverted to the natural amino acid found in wild-type trypsin without affecting the favorable synthetic properties obtained by the newly introduced mutation at the random position.

[0113] Table 6: Summary of enzymatic parameters for Trypsiligase II, A2C8, and K7F11. Product yields and apparent turnover rates of aminolysis using two equivalents of the corresponding nucleophile at high and low substrate concentrations in a model transamidation reaction. and the apparent turnover rate of hydrolysis Reaction conditions for A2C8 and K7F11: 250 / 15 μM Bz-PGGYRKKAG-OH (acyl donor), 500 / 30 μM H-RKKAK-OH (acyl acceptor), 0.75-2 μM trypsin variants, 100 mM HEPES / NaOH pH 7.8, 100 mM NaCl, 10 mM CaCl2, T = 30°C. Reaction conditions for Trypsiligase II: 250 / 15 μM Bz-PGGYRAHAG-OH (acyl donor), 500 / 30 μM H-RAHAK-OH (acyl acceptor), 0.75-2.5 μM trypsin variants, 100 mM HEPES / NaOH pH 7.8, 0.1 mM ZnCl2, 100 mM NaCl, 10 mM CaCl2, T = 30°C. UPLC analysis: Waters Acquity Ultra Performance LC, C18 column, gradient 5-40% acetonitrile over 4 minutes, detection at 254 nm.

[0114]

[0115] The suitability of variants A2C8 and K7F11 as transamidases was further investigated in comparison with native Trypsiligase II. Therefore, the enzymatic parameters for catalyzing transamidation reactions were determined through model transamidation reactions using peptide substrates with preferred recognition motifs. Furthermore, model transamidation reactions were performed at high (250 μM) and low (15 μM) substrate concentrations to test whether the new biocatalysts could also efficiently catalyze the desired reaction at low substrate concentrations, a key requirement for therapeutic protein modification. At high substrate concentration, native Trypsiligase II displayed only a poor aminolysis-to-hydrolysis ratio of 0.9, resulting in a moderate product yield of 17%. Lowering the substrate concentration to 15 μM resulted in a significant decrease in the apparent turnover rate of the aminolysis reaction, while the deacylation step was dominated by the hydrolysis reaction, which had a tenfold higher turnover rate, resulting in a poor product yield of 3%. The improved biocatalysts displayed significantly improved synthesis efficiency, especially variant A2C8. At a substrate concentration of 250 μM, A2C8 exhibited an excellent aminolysis to hydrolysis ratio of 70, with a final product yield of 64.9%, nearly matching the theoretically achievable yield of 67%, which is thermodynamically limited under the given reaction conditions (two-fold excess of nucleophile). Even at low substrate concentrations, the aminolysis reaction clearly dominated the deacylation step, exhibiting an apparent turnover rate seven times higher than that of the hydrolysis reaction. With a product yield of 46%, A2C8 exhibited a 14-fold higher product yield than native Trypsiligase II. It is assumed that the improved transamidation activity was achieved by introducing mutations that improved the biocatalyst's affinity for the nucleophilic peptide and / or reduced its hydrolytic activity. Accordingly, corresponding experimental data describing parameters for both native Trypsiligase II and variant A2C8 were determined. A summary of these data is listed in Table 7 and includes the enzyme's K for the nucleophilic peptide. M values ​​and the turnover rate of hydrolysis in the absence of nucleophilic peptides.

[0116] The first indication that the putative increased affinity for nucleophilic peptides already applied to native Trypsiligase II was observed in studies of the zinc dependence of the transamidation reaction catalyzed by Trypsiligase II. It was shown that Trypsiligase II is strongly dependent on zinc ions, as its absence leads to a significant decrease in the apparent turnover rate of the aminolysis reaction and, more importantly, to a significant reduction in product yield by a factor of 4. As described above, the reduced enzymatic affinity for nucleophilic peptides is due to the lack of zinc-mediated complexation between the artificial histidines (H39 and H59) of Trypsiligase II and the peptide-localized histidine in the P3' position.

[0117] By measuring the K of nucleophilic peptides in the presence or absence of zinc ions MThe values ​​confirmed the zinc-dependent effect on the affinity of trypsiligase II. In the presence of zinc ions, the K M The value is 143 μM. In the absence of zinc ions, the K M The value increased 10-fold to 1630 μM. Under the same substrate and reaction conditions, the product yield of variant A2C8 reached 59.7% independently of the presence of zinc ions. A key element of this enhanced transamidation reaction is based on the further improved enzyme affinity for nucleophilic peptides. In the absence of zinc ions, A2C8 has a K of 17.6 μM for nucleophilic peptides. M Compared to native Trypsiligase II, this corresponds to an increase in the enzyme affinity for the nucleophilic peptide by a factor of 8 and 92 in the presence or absence of zinc ions, respectively.

[0118] Another key element in the enhanced transamidation reaction of A2C8 relies on its reduced intrinsic hydrolytic activity compared to native Trypsiligase II. In the absence of a nucleophilic peptide, A2C8 exhibited a hydrolysis turnover rate of 86.6 mkat / mol, while Trypsiligase II's turnover rate was determined to be 602.1 mkat / mol. The enzyme's hydrolytic activity, which was reduced by a factor of 7, is an important parameter because hydrolysis competes with aminolysis in the deacylation step, thereby determining the product yield of the synthetic reaction. In summary, we demonstrate that the enhanced transamidation performance of A2C8 is due to both improved enzyme affinity for the nucleophilic peptide and reduced intrinsic hydrolytic activity.

[0119] These two features of A2C8 are directly related to the new mutations introduced into Trypsiligase II by our evolutionary approach. Table 7: Summary of enzymatic parameters of Trypsiligase II and the improved variant A2C8. Turnover rate (k) of the hydrolysis reaction with Bz-PGGYRAHAG-OH as the acyl donor cat,HL ) and the K of nucleophilic peptides (acyl acceptors) M The K of the acyl acceptor was determined by measuring the apparent turnover rate of the corresponding aminolysis reaction at a constant acyl donor concentration and a varying acyl acceptor concentration. M For Trypsiligase II, due to its strong dependence on zinc ions, the K MThe values ​​were measured in the presence or absence of zinc ions. The hydrolysis reaction was carried out under the following conditions: varying concentrations of Bz-PGGYRAHAG-OH (acyl donor), 2 μM trypsin variant, 100 mM HEPES / NaOH pH 7.8, ±0.1 mM ZnCl2, 100 mM NaCl, 10 mM CaCl2, T = 30°C. UPLC analysis conditions: Waters Acquity Ultra Performance LC, C18 column, gradient 5%-40% acetonitrile over 5 minutes, detection at 254 nm; K M The transamidation reaction was performed under the following conditions: 250 μM Bz-PGGYRAHAG-OH (acyl donor), varying concentrations of H-RAHAK(DNP)-OH (acyl acceptor), 0.2-1.5 μM trypsin variants, 100 mM HEPES / NaOH pH 7.8, ±0.1 mM ZnCl2, 100 mM NaCl, 10 mM CaCl2, T = 30°C. Two equivalents of acyl acceptor and 250 μM acyl donor compound were used to determine the maximum product yield. UPLC analysis conditions: Waters Acquity Ultra Performance LC, C18 column, gradient 5-60% acetonitrile over 5 minutes, detection at 360 nm.

[0120]

[0121] Example 5

[0122] To demonstrate the activating effect of the A2C8 and K7F11 mutations introduced by evolutionary selection, in the next step, the mutations Y39H, Y59H, K60E, and D189K derived from Trypsiligase II were gradually mutated back to the amino acid residues present in wild-type trypsin, and the synthetic potential was analyzed. These data show that even without mutations derived from Trypsiligase II (Y39H / Y59H / K60E / D189K) or Trypsiligase I (K60E / N143H / E151H / D189K), the positions to be protected by this patent are sufficient to induce the transamidation activity of wild-type trypsin.

[0123] As a first step, the effect of backmutations of artificial histidines at positions 39 and 59 in A2C8 to tyrosines (where wild-type trypsin finds tyrosines) was investigated by model transamidation reactions. Variants with a single backmutation at position 39 or 59 (A2C8_H39Y and A2C8_H59Y) as well as a variant with mutations at both positions 39 and 59 (A2C8_H39Y / H59Y) were investigated. Figure 3 ).

[0124] Variants with a single back mutation at position 39 or 59 (A2C8_H39Y and A2C8_H59Y) and a variant with mutations at both positions 39 and 59 (A2C8_H39Y / H59Y) showed comparable synthetic performance to variant A2C8 (e.g. Figure 3 This leads to the conclusion that the wild-type mutations at positions 39 and 59 have no effect on the synthesis efficiency of A2C8, indicating that the favorable synthesis properties of A2C8 are obtained by newly introduced mutations at random positions.

[0125] To further investigate whether the identified mutations were sufficient to convert the wild-type protease trypsin into a transamidase, two additional Trypsiligase II related mutations at positions 189 and 60 were mutated back to the wild-type amino acids. Reversion mutations at positions 189 and 60 were introduced into the variant A2C8_H39Y / H59Y as single reversion mutations (A2C8_H39Y / H59Y / E60K and A2C8H39Y / H59Y / K189D) as well as double mutations (A2C8_H39Y / H59Y / E60K / K189D). In a first step, the substrate specificity of all variants was investigated, since position 189 is known to affect the S1 specificity of trypsin and position 60 is known to affect the S1' specificity ( Figure 4 ).

[0126] The back mutation at position 60 in variant A2C8_H39Y / H59Y / E60K causes greater flexibility at the P1' position of the substrate, thereby also allowing acceptance of methionine or alanine. The back mutation at position 189 in variant A2C8_H39Y / H59Y / K189D causes greater flexibility at the P1 position of the substrate, thereby also allowing acceptance of arginine, which is related to the specificity of wild-type trypsin. The variant A2C8_H39Y / H59Y / E60K / K189D with all four back mutations shows a specificity spectrum that combines the flexibility of A2C8_H39Y / H59Y / E60K at the P1' position of the substrate and the flexibility of A2C8_H39Y / H59Y / K189E at the P1 position of the substrate.

[0127] The three substrates with the highest apparent turnover rates for the aminolysis reaction were then used to investigate the synthetic performance of variant A2C8_H39Y / H59Y / E60K / K189D. Figure 5 In FIG, the time course of product formation of the transamidation reaction catalyzed by the variant A2C8_H39Y / H59Y / E60K / K189D using various peptide substrates is shown.

[0128] like Figure 5As shown, variant A2C8_H39Y / H59Y / E60K / K189D can effectively catalyze the formation of transamidation products using substrates with the recognition sequences YRRH, YMKH, and RMKH. The highest product yield of 38% was observed for substrates with the recognition sequence YRRH. Under comparable conditions, the product yield of A2C8 reached 59%. For wild-type trypsin, almost no product formation was observed. This confirms the previous assumption that the newly introduced mutations are sufficient to convert the wild-type protease trypsin into a transamidation enzyme.

[0129] Since these mutations are associated with an improvement in the enzyme affinity for nucleophilic peptides and a decrease in intrinsic hydrolytic activity, it can be concluded that all trypsin species can be converted into transaminases in general by introducing amino acid exchanges that affect an improvement in the enzyme affinity for nucleophilic peptides and / or a decrease in intrinsic hydrolytic activity.

[0130] Example 6

[0131] Subsequently, dual modification of the Fab fragment was investigated by exploiting the changes in substrate specificity of the two Trypsiligase II variants.

[0132] Interestingly, backmutation of the Trypsiligase II-associated mutations at positions 39, 59, and 189 within variant K7F11 (resulting in variant K7F11_H39Y / H59Y / K189D) resulted in a transamidase with alternating specificity at the P1 position.

[0133] K7F11_H39Y / H59Y / K189D has high specific activity towards the recognition sequence RRKH, whereas aromatic or aliphatic substitutions (accepted by at least A2C8 and K7F11) lead to a significant decrease in the apparent turnover rate of the aminolysis reaction.

[0134] This means that the variant K7F11_H39Y / H59Y / K189D is able to effectively discriminate the recognition sequence RRKH even in the presence of A2C8-related recognition sequences such as YRAH.

[0135] To demonstrate the feasibility of orthogonal dual modification of proteins at two different modification sites, a Her2-specific Fab fragment was constructed with an RRKH motif at the C-terminus of the light chain and a YRAH motif at the C-terminus of the heavy chain (see Figure 6 ).

[0136] Then perform the sequential double modification:

[0137] In the first step, the light chain was modified with a nucleophile containing carboxyfluorescein, catalyzed by variant K7F11_H39Y / H59Y / K189D. Mass spectrometry analysis revealed an almost exclusive modification at the C-terminal end of the light chain. The enzyme and the remaining nucleophile (RKHAK(CF)-OH) were removed by protein G affinity chromatography.

[0138] In the second step, the heavy chain was modified with a nucleophile carrying DM1, which was catalyzed by variant A2C8. The proprietary modification of the heavy chain C-terminus was confirmed by mass spectrometry. As estimated by mass spectrometry, the yield of the dual-labeled Fab fragment was approximately 75%.

[0139] In the case of orthogonal double modification, the term "orthogonal" refers to the use of two different biocatalysts from the same source to modify the polypeptide at two different recognition sequences without significant cross-reactivity, following a modification strategy that includes both N-terminal and C-terminal positioning of the recognition sequences (see Table 8).

[0140] Table 8: Summary of feasible enzyme and recognition sequence pairs within the context of dual and orthogonal modification of proteins.

[0141]

[0142] Table 9 below summarizes the most important key data of the relevant trypsiligase variants or libraries:

[0143] Table 9: Summary of data for the trypsiligase variants or libraries described herein.

[0144]

[0145]

[0146] The following sequences are disclosed herein:

[0147] Rat anionic trypsin II (SEQ ID NO: 1):

[0148]

[0149] Anti-Her2-Fab-LC_RRKH / HC_YRAH, heavy chain (SEQ ID NO: 2):

[0150]

[0151] Anti-Her2-Fab-LC_RRKH / HC_YRAH, light chain (SEQ ID NO: 3):

[0152]

[0153] The following polypeptides are further disclosed:

[0154]

[0155]

[0156]

[0157] DNP-2,4-dinitrophenyl

[0158] CF-5(6)-carboxyfluorescein

[0159] HexMal-6-Maleimidocaproic acid

[0160] PEG20.000 - polyethylene glycol, average mass 20000 g / mol

[0161] MCC-4-(N-maleimidomethyl)cyclohexane-1-carboxylate

[0162] Ac-acetate linker

[0163] Gene sequence of anti-Her2-Fab-LC_RRKH / HC_YRAH (SEQ ID NO: 64):

[0164]

[0165] Sequence Listing <110> Biopharmaceutical Translation Institute Dessau Research GmbH <120> Trypsin variants with improved enzymatic properties <130> F-CF210272 <150> EP18214031.9 <151> 2018-12-19 <160> 64 <170> SIPOSequenceListing 1.0 <210> 1 <211> 223 <212> PRT <213> Artificial Sequence <400> 1 Ile Val Gly Gly Tyr Thr Cys Gln Glu Asn Ser Val Pro Tyr Gln Val 1 5 10 15 Ser Leu Asn Ser Gly Tyr His Phe Cys Gly Gly Ser Leu Ile Asn Asp 20 25 30 Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Arg Ile Gln Val 35 40 45 Arg Leu Gly Glu His Asn Ile Asn Val Leu Glu Gly Asn Glu Gln Phe 50 55 60 Val Asn Ala Ala Lys Ile Ile Lys His Pro Asn Phe Asp Arg Lys Thr 65 70 75 80 Leu Asn Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Pro Val Lys Leu 85 90 95 Asn Ala Arg Val Ala Thr Val Ala Leu Pro Ser Ser Cys Ala Pro Ala 100 105 110 Gly Thr Gln Cys Leu Ile Ser Gly Trp Gly Asn Thr Leu Ser Ser Gly 115 120 125 Val Asn Glu Pro Asp Leu Leu Gln Cys Leu Asp Ala Pro Leu Leu Pro 130 135 140 Gln Ala Asp Cys Glu Ala Ser Tyr Pro Gly Lys Ile Thr Asp Asn Met 145 150 155 160 Val Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ser Cys Gln Gly Asp 165 170 175 Ser Gly Gly Pro Val Val Cys Asn Gly Glu Leu Gln Gly Ile Val Ser 180 185 190 Trp Gly Tyr Gly Cys Ala Leu Pro Asp Asn Pro Gly Val Tyr Thr Lys 195 200 205 Val Cys Asn Tyr Val Asp Trp Ile Gln Asp Thr Ile Ala Ala Asn 210 215 220 <210> 2[[ID=#15]] <211> 245 <212> PRT <213> Artificial Sequence <400> 2 Glu Val Lys Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15[[ID=##27]] Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Note: There are two tags in the original text that seem to have some non-standard formatting (tags with '#' and '##' in them). I've left them as they are in the translation as per the instruction to preserve all 7 - digit tags exactly as - is. It's possible there might be an error in the original tag formatting. 85 90 95 Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Ala 210 215 220 Asp Lys Pro Gly Gly Tyr Arg Ala His Ala Gly Glu Gln Lys Leu Ile 225 230 235 240 Ser Glu Glu Asp Leu 245 <210> 3 <211> 225 <212> PRT <213> Artificial Sequence <400> 3 Asp Ile Glu Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys Leu Ser Pro Gly Gly Arg Arg Lys His Ala 210 215 220 Gly 225 <210> 4 <211> 9 <212> PRT <213> Artificial Sequence <400> 4 Pro Gly Gly Tyr Arg Ala His Ala Gly 1 5 <210> 5 <211> 9 <212> PRT <213> Artificial Sequence <400> 5 Pro Gly Gly Phe Arg Ala His Ala Gly 1 5 <210> 6 <211> 9 <212> PRT <213> Artificial Sequence <400> 6 Pro Gly Gly Trp Arg Ala His Ala Gly 1 5 <210> 7 <211> 9 <212> PRT <213> Artificial Sequence <400> 7 Pro Gly Gly Leu Arg Ala His Ala Gly 1 5 <210> 8 <211> 9 <212> PRT <213> Artificial Sequence <400> 8 Pro Gly Gly Asp Arg Ala His Ala Gly 1 5 <210> 9 <211> 9 <212> PRT <213> Artificial Sequence <400> 9 Pro Gly Gly Arg Arg Ala His Ala Gly 1 5 <210> 10 <211> 9 <212> PRT <213> Artificial Sequence <400> 10 Pro Gly Gly Ala Arg Ala His Ala Gly 1 5 <210> 11 <211> 9 <212> PRT <213> Artificial Sequence <400> 11 Pro Gly Gly Tyr Ala Ala His Ala Gly 1 5 <210> 12 <211> 9 <212> PRT <213> Artificial Sequence <400> 12 Pro Gly Gly Tyr Asp Ala His Ala Gly 1 5 <210> 13 <211> 9 <212> PRT <213> Artificial Sequence <400> 13 Pro Gly Gly Tyr Glu Ala His Ala Gly 1 5 <210> 14 <211> 9 <212> PRT <213> Artificial Sequence <400> 14 Pro Gly Gly Tyr Lys Ala His Ala Gly 1 5 <210> 15 <211> 9 <212> PRT <213> Artificial Sequence <400> 15 Pro Gly Gly Tyr Arg Ala Ala Ala Gly 1 5 <210> 16 <211> 9 <212> PRT <213> Artificial Sequence <400> 16 Pro Gly Gly Tyr Arg Ala Asn Ala Gly 1 5 <210> 17 <211> 9 <212> PRT <213> Artificial Sequence <400> 17 Pro Gly Gly Tyr Arg Ala Lys Ala Gly 1 5 <210> 18 <211> 9 <212> PRT <213> Artificial Sequence <400> 18 Pro Gly Gly Tyr Arg Ala Asp Ala Gly 1 5 <210> 19 <211> 9 <212> PRT <213> Artificial Sequence <400> 19 Pro Gly Gly Tyr Arg Lys His Ala Gly 1 5 <210> 20 <211> 9 <212> PRT <213> Artificial Sequence <400> 20 Pro Gly Gly Phe Arg Lys His Ala Gly 1 5 <210> twenty one <211> 9 <212> PRT <213> Artificial Sequence <400> twenty one Pro Gly Gly Trp Arg Lys His Ala Gly 1 5 <210> twenty two <211> 9 <212> PRT <213> Artificial Sequence <400> twenty two Pro Gly Gly Leu Arg Lys His Ala Gly 1 5 <210> twenty three <211> 9 <212> PRT <213> Artificial Sequence <400> twenty three Pro Gly Gly Asp Arg Lys His Ala Gly 1 5 <210> twenty four <211> 9 <212> PRT <213> Artificial Sequence <400> twenty four Pro Gly Gly Arg Arg Lys His Ala Gly 1 5 <210> 25 <211> 9 <212> PRT <213> Artificial Sequence <400> 25 Pro Gly Gly Ala Arg Lys His Ala Gly 1 5 <210> 26 <211> 9 <212> PRT <213> Artificial Sequence <400> 26 Pro Gly Gly Tyr Ala Lys His Ala Gly 1 5 <210> 27 <211> 9 <212> PRT <213> Artificial Sequence <400> 27 Pro Gly Gly Tyr Asp Lys His Ala Gly 1 5 <210> 28 <211> 9 <212> PRT <213> Artificial Sequence <400> 28 Pro Gly Gly Tyr Met Lys His Ala Gly 1 5 <210> 29 <211> 9 <212> PRT <213> Artificial Sequence <400> 29 Pro Gly Gly Tyr Arg Arg His Ala Gly 1 5 <210> 30 <211> 9 <212> PRT <213> Artificial Sequence <400> 30 Pro Gly Gly Tyr Arg Lys Ala Ala Gly 1 5 <210> 31 <211> 9 <212> PRT <213> Artificial Sequence <400> 31 Pro Gly Gly Tyr Arg Lys Asn Ala Gly 1 5 <210> 32 <211> 9 <212> PRT <213> Artificial Sequence <400> 32 Pro Gly Gly Tyr Arg Lys Lys Ala Gly 1 5 <210> 33 <211> 9 <212> PRT <213> Artificial Sequence <400> 33 Pro Gly Gly Tyr Arg Lys Asp Ala Gly 1 5 <210> 34 <211> 9 <212> PRT <213> Artificial Sequence <400> 34 Pro Gly Gly Tyr Lys Arg Lys Ala Gly 1 5 <210> 35 <211> 9 <212> PRT <213> Artificial Sequence <400> 35 Pro Gly Gly Arg Met Lys His Ala Gly 1 5 <210> 36 <211> 5 <212> PRT <213> Artificial Sequence <400> 36 Arg Ala His Ala Lys 1 5 <210> 37 <211> 5 <212> PRT <213> Artificial Sequence <400> 37 Ala Ala His Ala Lys 1 5 <210> 38 <211> 5 <212> PRT <213> Artificial Sequence <400> 38 Asp Ala His Ala Lys 1 5 <210> 39 <211> 5 <212> PRT <213> Artificial Sequence <400> 39 Glu Ala His Ala Lys 1 5 <210> 40 <211> 5 <212> PRT <213> Artificial Sequence <400> 40 Lys Ala His Ala Lys 1 5 <210> 41 <211> 5 <212> PRT <213> Artificial Sequence <400> 41 Arg Ala Ala Ala Lys 1 5 <210> 42 <211> 5 <212> PRT <213> Artificial Sequence <400> 42 Arg Ala Asn Ala Lys 1 5 <210> 43 <211> 5 <212> PRT <213> Artificial Sequence <400> 43 Arg Ala Lys Ala Lys 1 5 <210> 44 <211> 5 <212> PRT <213> Artificial Sequence <400> 44 Arg Ala Asp Ala Lys 1 5 <210> 45 <211> 5 <212> PRT <213> Artificial Sequence <400> 45 Arg Lys His Ala Lys 1 5 <210> 46 <211> 5 <212> PRT <213> Artificial Sequence <400> 46 Ala Lys His Ala Lys 1 5 <210> 47 <211> 5 <212> PRT <213> Artificial Sequence <400> 47 Asp Lys His Ala Lys 1 5 <210> 48 <211> 5 <212> PRT <213> Artificial Sequence <400> 48 Met Lys His Ala Lys 1 5 <210> 49 <211> 5 <212> PRT <213> Artificial Sequence <400> 49 Arg Arg His Ala Lys 1 5 <210> 50 <211> 5 <212> PRT <213> Artificial Sequence <400> 50 Arg Lys Ala Ala Lys 1 5 <210> 51 <211> 5 <212> PRT <213> Artificial Sequence <400> 51 Arg Lys Asn Ala Lys 1 5 <210> 52 <211> 5 <212> PRT <213> Artificial Sequence <400> 52 Arg Lys Lys Ala Lys 1 5 <210> 53 <211> 5 <212> PRT <213> Artificial Sequence <400> 53 Arg Lys Asp Ala Lys 1 5 <210> 54 <211> 5 <212> PRT <213> Artificial Sequence <400> 54 Lys Arg Lys Ala Lys 1 5 <210> 55 <211> 5 <212> PRT <213> Artificial Sequence <400> 55 Arg Ala His Ala Lys 1 5 <210> 56 <211> 5 <212> PRT <213> Artificial Sequence <400> 56 Arg Lys His Ala Lys 1 5 <210> 57 <211> 5 <212> PRT <213> Artificial Sequence <400> 57 Arg Lys Lys Ala Lys 1 5 <210> 58 <211> 5 <212> PRT <213> Artificial Sequence <400> 58 Arg Lys Ala Ala Lys 1 5 <210> 59 <211> 5 <212> PRT <213> Artificial Sequence <400> 59 Lys Arg Lys Ala Lys 1 5 <210> 60 <211> 5 <212> PRT <213> Artificial Sequence <400> 60 Arg Light His Wing Light 1 5 <210> 61 <211> 8 <212> PRT <213> Artificial Sequence <400> 61 Arg Light Light Sky Light Sky Light 1 5 <210> 62 <211> 5 <212> PRT <213> Artificial Sequence <400> 62 Arg Light Light Sky Light 1 5 <210> 63 <211> 5 <212> PRT <213> Artificial Sequence <400> 63 Arg Light Light Ala Light 1 5 <210> 64 <211> 1552 <212> DNA <213> Artificial Sequence <400> 64 atgaagaaaa ccgcgattgc gattgcggtg gcgctggcgg gctttgcgac cgtggcgcag gcggatattg aactgaccca gagcccgagc agcctgagcg cgagcgtggg cgatcgcgtg 120 accattacct gccgcgcgag ccaggatgtg aacaccgcgg tggcgtggta tcagcagaaa 180 ccgggcaaag cgccgaaact gctgatttat agcgcgagct ttctgtatag cggcgtgccg 240 agccgcttta gcggcagccg cagcggcacc gattttaccc tgaccattag cagcctgcag 300 ccggagatt ttgcgaccta ttattgccag cagcattata ccccccgcc gaccttttggc 420. cagggcacca aactggaaat taaacgcacc gtggcggcgc cgagcgtgtt tattttccg ccgagcgatg aacagctgaa aagcggcacc gcgagcgtgg tgtgcctgct gaacaacttt tatccgcgcg aagcgaaagt gcagtggaaa gtggataacg cgctgcagag cggcaacagc 540 caggaaagcg tgaccgaaca ggatagcaaa gatagcacct atagcctgag cagcaccctg accctgagca aagcggatta tgaaaaacat aaagtgtatg cgtgcgaagt gacccatcag ggcctgagca gcccggtgac caaatctttt aaccgcggcg aatgcctgag ccccggagga 720 cgccgcaaac atgcgggctg aggaggaaaa aaaaatgaaa aagacagcta tcgcaattgc agtggcgcta gctggtttcg ccaccgtggc gcaagctgaa gtgaaactgc aggaaagcgg 840 tggtggtctg gtgcagccgg gtggtagcct gcgcctgagc tgcgcggcga gcggctttaa900 cattaagat acctatattc attgggtgcg ccaggcgccg ggcaaaggcc tggaatgggt ggcgcgcatt tatccgacca acggctatac ccgctatgcg gatagcgtga aaggccgctt 1080. gcggatacca gcaaaaacac cgcgtatctg cagatgaaca gcctgcgcgc ggaagatacc gcggtgtatt attgcagccg ctggggcggc gatggctttt atgcgatgga 1140 ttattggggc cagggcaccc tggtgaccgt gagcagcgcg agcaccaaag gcccgagcgt gtttccgctg gcgccgagca gcaaaagcac cagcggcggc accgcggcgc tgggctgcct 1260 ggtgaaagat tattttccgg aaccggtgac cgtgagctgg aacagcggcg cgctgaccag cggcgtgcat acctttccgg cggtgctgca gagcagcggc ctgtatagcc tgagcagcgt ggtgaccgtg ccgagcagca gcctgggcac ccagacctat atttgcaacg tgaaccataa accgagcaac accaaagtgg ataaaaagt ggaccgaa agctgcgcgg ataaacccgg aggatatcgc gcgcatgcgg gcgaacagaa actgattagc gaagaatc tg

Claims

1. A mutant trypsin comprising amino acid substitutions at at least two amino acid positions that result in increased affinity for nucleophilic substrates and / or amino acid substitutions at at least two amino acid positions that result in decreased hydrolytic activity, wherein the amino acid substitutions of the mutant trypsin are: (1) Y39H, Y59H, K60E, D189K, H40F, A55A, R96E, K97D, L99F, N143E, E151Y, S190V, Q192A, S214G, G219Q, and A221T; (2) Y59H, K60E, D189K, H40F, A55A, R96E, K97D, L99F, N143E, E151Y, S190V, Q192A, S214G, G219Q, and A221T; (3) Y39H, K60E, D189K, H40F, A55A, R96E, K97D, L99F, N143E, E151Y, S190V, Q192A, S214G, G219Q, and A221T; (4) K60E, D189K, H40F, A55A, R96E, K97D, L99F, N143E, E151Y, S190V, Q192A, S214G, G219Q, and A221T; (5) D189K, H40F, A55A, R96E, K97D, L99F, N143E, E151Y, S190V, Q192A, S214G, G219Q, and A221T; (6) K60E, H40F, A55A, R96E, K97D, L99F, N143E, E151Y, S190V, Q192A, S214G, G219Q, and A221T; (7) H40F, A55A, R96E, K97D, L99F, N143E, E151Y, S190V, Q192A, S214G, G219Q, and A221T; (8) Y39H, Y59H, K60E, D189K, H40Y, A55A, R96E, K97E, L99F, N143V, E151E, S190A, Q192V, S214G, G219P, and A221Q; (9) Y59H, K60E, D189K, H40Y, A55A, R96E, K97E, L99F, N143V, E151E, S190A, Q192V, S214G, G219P, and A221Q; (10) Y39H, K60E, D189K, H40Y, A55A, R96E, K97E, L99F, N143V, E151E, S190A, Q192V, S214G, G219P, and A221Q; (11) K60E, D189K, H40Y, A55A, R96E, K97E, L99F, N143V, E151E, S190A, Q192V, S214G, G219P, and A221Q; (12) D189K, H40Y, A55A, R96E, K97E, L99F, N143V, E151E, S190A, Q192V, S214G, G219P, and A221Q; (13) K60E, H40Y, A55A, R96E, K97E, L99F, N143V, E151E, S190A, Q192V, S214G, G219P, and A221Q; or (14) H40Y, A55A, R96E, K97E, L99F, N143V, E151E, S190A, Q192V, S214G, G219P, and A221Q; in, The wild type of the mutated trypsin is the amino acid sequence shown in SEQ ID NO:

1. According to the chymotrypsin nomenclature, Y39, H40, A55, Y59, K60, R96, K97, L99, N143, E151, D189, S190, Q192, S214, G219, and A221 correspond to positions 22, 23, 38, 42, 43, 78, 79, 81, 123, 131, 171, 172, 174, 192, 196, and 198 of the sequence of mature anionic rat trypsin II from Rattus norvegicus as shown in SEQ ID NO: 1, respectively.

2. Use of two different trypsins for orthogonal double modification of two different recognition sequences, wherein the first enzyme is trypsin variant A2C8 and the second enzyme is trypsin variant K7F11, or wherein the first enzyme is trypsin variant A2C8 and the second enzyme is trypsin variant K7F11_H39Y / H59Y / K189D, or wherein the first enzyme is Trypsiligase II and the second enzyme is trypsin variant A2C8, or wherein the first enzyme is Trypsiligase II and the second enzyme is trypsin variant K7F11, or wherein the first enzyme is trypsin variant K7F11_H39Y / H59Y / K189D and the second enzyme is trypsin variant A2C8, or wherein the first enzyme is trypsin variant K7F11_H39Y / H59Y / K189D and the second enzyme is trypsin variant K7F11, wherein the amino acid substitutions of the trypsin variant A2C8 are: Y39H, Y59H, K60E, D189K, H40F, A55A, R96E, K97D, L99F, N143E, E151Y, S190V, Q192A, S214G, G219Q and A221T, The amino acid substitutions of the trypsin variant K7F11 are: Y39H, Y59H, K60E, D189K, H40Y, A55A, R96E, K97E, L99F, N143V, E151E, S190A, Q192V, S214G, G219P and A221Q, The amino acid substitutions of the trypsin variant K7F11_H39Y / H59Y / K189D are: K60E, H40Y, A55A, R96E, K97E, L99F, N143V, E151E, S190A, Q192V, S214G, G219P and A221Q, The amino acid substitutions of the Trypsiligase II are: Y39H, Y59H, K60E and D189K, in, The wild type of trypsin is the amino acid sequence shown in SEQ ID NO:

1. According to the chymotrypsin nomenclature, Y39, H40, A55, Y59, K60, R96, K97, L99, N143, E151, D189, S190, Q192, S214, G219, and A221 correspond to positions 22, 23, 38, 42, 43, 78, 79, 81, 123, 131, 171, 172, 174, 192, 196, and 198 of the sequence of mature anionic rat trypsin II from Rattus norvegicus as shown in SEQ ID NO:1, respectively.

3. A method for orthogonal double modification of a substrate, comprising the following steps: a) providing an orthogonal double-modified substrate, b) modifying the substrate using a first trypsin that recognizes a first recognition sequence, c) modifying the substrate using a second trypsin that recognizes a second recognition sequence, wherein the first trypsin or the second trypsin is selected from the group consisting of trypsin variant A2C8, trypsin variant K7F11, and trypsin variant K7F11_H39Y / H59Y / K189D, wherein the amino acid substitutions of the trypsin variant A2C8 are: Y39H, Y59H, K60E, D189K, H40F, A55A, R96E, K97D, L99F, N143E, E151Y, S190V, Q192A, S214G, G219Q and A221T, The amino acid substitutions of the trypsin variant K7F11 are: Y39H, Y59H, K60E, D189K, H40Y, A55A, R96E, K97E, L99F, N143V, E151E, S190A, Q192V, S214G, G219P and A221Q, The amino acid substitutions of the trypsin variant K7F11_H39Y / H59Y / K189D are: K60E, H40Y, A55A, R96E, K97E, L99F, N143V, E151E, S190A, Q192V, S214G, G219P and A221Q, Wherein, the wild type of the first trypsin and the second trypsin is the amino acid sequence shown in SEQ ID NO:1, and according to the chymotrypsin nomenclature, Y39, H40, A55, Y59, K60, R96, K97, L99, N143, E151, D189, S190, Q192, S214, G219, and A221 correspond to positions 22, 23, 38, 42, 43, 78, 79, 81, 123, 131, 171, 172, 174, 192, 196, and 198 of the sequence of mature anionic rat trypsin II from Rattus norvegicus as shown in SEQ ID NO:1, respectively.

Images