Sortase having improved thermal stability

Abstract

The present application relates to a sortase mutant having improved catalytic activity and thermal stability, and also relates to the use of the sortase mutant.

Description

Sortase with improved thermal stability TECHNICAL FIELD

[0001] The present application relates to sortase variants with improved catalytic activity and thermal stability. The present application also relates to the use of said sortase mutants. BACKGROUND

[0002] Bacterial sortases are transpeptidases that can modify proteins in a site-specific manner. The most widely used sortase is the wild-type sortase A (SrtA) derived from Staphylococcus aureus, which forms an enzyme-substrate (thioester) intermediate by recognizing the motif LPXTG and cleaving the peptide bond between threonine and glycine, followed by the disassembly of the intermediate by nucleophilic attack, resulting in the formation of a new peptide bond between the substrate and the target, thus enabling labeling or conjugation. Therefore, sortases are widely used in protein engineering, for example, in terms of site-directed modification and site-directed ligation. Wild-type SrtA contains 206 amino acid residues, and due to the recognition of a specific motif, it results in a limited substrate spectrum. For this reason, the field has been working to develop sortase mutants with desired properties.

[0003] However, existing sortase mutants have the problem of poor thermal stability during the production and purification process. Usually, purification needs to be carried out in a low-temperature environment of 2-8°C. When purification is carried out at room temperature, the sortase will easily aggregate and degrade, resulting in very low enzyme activity of the final product. The inability of sortase to be purified at room temperature increases the difficulty and cost of its industrial scale production. In addition, at a higher temperature (such as 40°C), the enzyme activity of sortase will also be significantly reduced if left for a long time.

[0004] Red blood cells (RBCs) have been considered as ideal in vivo drug delivery systems due to their unique biological properties (e.g. long in vivo survival time, low immunogenicity, good biocompatibility, etc.). Various attempts have been made to develop red blood cells that can serve as drug delivery carriers, for example, by means of sortase to site-direct drugs to the surface of the red blood cell membrane. Therefore, the thermal stability of sortase has become one of the problems that restrict the engineering directed modification and industrial production application of red blood cells. For this reason, there is still an urgent need in the field to improve or enhance the activity and thermal stability of sortase.

[0005] The present application provides sortases with improved performance, which to some extent solves the above-mentioned problem.

[0006] SUMMARY

[0007] In a first aspect, the present application provides sortase mutants, which have the following advantages:

[0008] 1. excellent catalytic activity;

[0009] 2. Improved thermal stability;

[0010] 3. Efficient recognition of receptor motifs comprising only 1 glycine at the N-terminus;

[0011] 4. Efficient recognition of receptor motifs comprising 3 glycines at the N-terminus;

[0012] 5. Efficiently promote the directed coupling of specific proteins, drug molecules to cells (e.g. red blood cells), and increase the loading capacity of the cells.

[0013] In some embodiments, the present application provides a sortase mutant having an amino acid sequence which is at least 90% identical to a parent sortase as set forth in SEQ ID NO: 2, and comprising one or more mutations selected from P94R, D124G, M155V, D160N, K162P, Y187L, E189R, K190E, and F200L (numbering according to the sequence set forth in SEQ ID NO: 1).

[0014] In some specific embodiments, the sortase mutant has an amino acid sequence which is at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, or at least 96% identical to a parent sortase as set forth in SEQ ID NO: 2, and comprises the mutations D124G / M155V / Y187L / E189R / K190E, D124G / M155V / D160N / Y187L / E189R, P94R / D124G / M155V / Y187L / E189R, or P94R / D124G / M155V / K162P / Y187L / E189R / F200L.

[0015] In some specific embodiments, the sortase mutant comprises or consists of the amino acid sequence set forth in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6.

[0016] In a second aspect, the present application provides a polynucleotide molecule encoding the sortase mutant of the first aspect.

[0017] In a third aspect, the present application provides a vector comprising the polynucleotide molecule of the second aspect.

[0018] In a fourth aspect, the present application provides a cell comprising the polynucleotide molecule of the second aspect or the vector of the third aspect. In some specific embodiments, the cell is a eukaryotic cell.

[0019] In a fifth aspect, the present application provides a method for coupling an active molecule to a cell, comprising contacting the sortase mutant of the first aspect with the cell under conditions suitable for the active molecule to be coupled to the cell by the sortase mutant.

[0020] In some embodiments, the cell is a human-derived cell, such as a PBMC cell. In some embodiments, the human cell is a T cell, a NK cell, a macrophage cell, a DC cell, a B cell. In some embodiments, the human cell is a red blood cell.

[0021] In some specific embodiments, the cell membrane surface of the human cell (e.g. a red blood cell) comprises a sortase receptor motif. In other specific embodiments, the cell membrane surface of the human cell (e.g. a red blood cell) comprises a linker, which comprises a sortase receptor motif. In other specific embodiments, the sortase receptor motif is, for example, a small peptide comprising one or more glycines, such as a small peptide comprising one glycine or three glycines, such as a receptor motif comprising the sequence set forth in SEQ ID NO: 7. In other specific embodiments, the active molecule comprises a sortase recognition motif. In other specific embodiments, the sortase recognition motif is, for example, the amino acid sequence LPXTG, LPETGK (SEQ ID NO: 13), LPETGGK (SEQ ID NO: 14).

[0022] In a sixth aspect, the present application provides the use of the sortase mutant of the first aspect in the directed coupling of molecules. In some specific embodiments, the present application provides the use of the sortase mutant of the first aspect in the directed coupling of an active molecule to the cell membrane surface of a human cell (e.g. a red blood cell). BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 shows the SDS-PAGE electrophoresis results of the product obtained after affinity chromatography (left panel) and cation exchange chromatography (right panel) of SrtA-V1.

[0024] Figure 2 shows the SDS-PAGE electrophoresis results of the product obtained after affinity chromatography (left panel) and cation exchange chromatography (right panel) of SrtA-V2.

[0025] Figure 3 shows the SDS-PAGE electrophoresis results of the product obtained after affinity chromatography (left panel) and cation exchange chromatography (right panel) of SrtA-V3.

[0026] Figure 4 shows the SDS-PAGE electrophoresis results of the product obtained after affinity chromatography (left panel) and cation exchange chromatography (right panel) of SrtA-V4.

[0027] Figure 5 shows the change rate of Vmax before and after heat treatment of each mutant

[0028] Figure 6 shows the DSC detection results of the parental sorting enzyme (SEQ ID NO:2) in different buffer solutions.

[0029] Figure 7 shows the DSC results of each mutant SrtA-V1, SrtA-V2, SrtA-V3 and SrtA-V4.

[0030] Figure 8 shows the time progression of in vitro transpeptidation catalyzed by each mutant under different NH2-G small peptide substrates, and the results of in vitro transpeptidation catalyzed by each mutant at different enzyme concentrations. (a) represents the time progression of transpeptidation catalyzed by each mutant at different reaction times; (b) represents the results of transpeptidation catalyzed by each mutant under different concentrations.

[0031] Figure 9 shows the three-dimensional protein structure of SrtA-V1 predicted by the Swiss-model.

[0032] Figure 10 shows the three-dimensional protein structure of SrtA-V2 predicted by the Swiss-model.

[0033] Figure 11 shows the three-dimensional protein structure of SrtA-V3 predicted by the Swiss-model.

[0034] Figure 12 shows the three-dimensional protein structure of SrtA-V4 predicted by the Swiss-model.

[0035] Figure 13 shows the flow cytometry results, indicating that the sorting enzyme mutant can catalyze the successful coupling of GFP-LPETG to the cell membranes of T cells, NK cells, macrophages, and B cells.

[0036] Invention Details

[0037] I. Definition

[0038] In this disclosure, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Any methods and materials similar to or equivalent to those described herein may be used to practice the invention, but preferred methods and materials are described herein. Therefore, the terminology defined herein is described more fully in connection with the specification as a whole. Furthermore, the terminology used herein is for illustrative purposes only and is not intended to be limiting.

[0039] As used herein, the singular terms "a," "an," and "the" encompass the plural unless the context clearly dictates otherwise. Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation. It is to be understood that this application is not limited to the particular methodology, protocols, and reagents described, as these can vary.

[0040] Unless the context requires otherwise, the terms "comprise," "comprises," and "comprising," or the like, are used on the basis and encompassing of a open-ended inclusion meaning that elements other than those listed after the word are optional and do not exclude additional elements.

[0041] As used herein, the term "sequence identity" refers to the number of nucleotides or amino acids that are exactly matched when the sequences are compared over a comparison window using a suitable alignment algorithm. Thus, the "percent sequence identity" is calculated by comparing two optimally aligned sequences over a comparison window, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to yield the percent sequence identity. For example, "sequence identity" can be understood to mean the "percent match" as calculated by the DNASIS computer program (Windows Version 2.5; available from Hitachi Software Engineering, Ltd., South San Francisco, Calif.).

[0042] In the context of polypeptides, the term "mutation" refers to the deletion, substitution, or insertion of at least one amino acid residue in a parent amino acid sequence. The replacement residue(s) can be "naturally occurring amino acid residues" (i.e., encoded by the genetic code) or "non-naturally occurring amino acid residues." Non-naturally occurring amino acid residues refer to residues other than naturally occurring amino acid residues that are capable of covalently bonding to one or more adjacent amino acid residues in a polypeptide chain, such as norleucine, ornithine, norvaline, homoserine, aib, and other amino acid residue analogs.

[0043] The term "amino acid deletion" refers to the removal of at least one amino acid residue at a predetermined position in an amino acid sequence.

[0044] The term "amino acid substitution" refers to the replacement of at least one amino acid residue in a predetermined parent amino acid sequence with a different amino acid residue. The replacement residue(s) can be "naturally occurring amino acid residues" (i.e., encoded by the genetic code) or "non-naturally occurring amino acid residues."

[0045] The term "position" in the context of a polypeptide refers to the position of an amino acid residue in the amino acid sequence of the polypeptide. In any case, positions are numbered sequentially, with the first amino acid residue being numbered 1.

[0046] The terms "individual" or "subject" are used interchangeably and refer to a mammal that can be treated using the methods or compositions disclosed herein. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). Thus, the methods and compositions disclosed herein can have medical and / or veterinary applications. In preferred forms, the mammal is a human.

[0047] The term "drug loading" refers to the amount of drug loaded per unit weight or unit volume or individual cell (e.g., red blood cell). In the present application, the drug loading of a cell (e.g., red blood cell) is usually measured in μg / mL as a dosage unit.

[0048] The term "treatment" refers to clinical intervention designed to alter the natural course of the disease in an individual receiving treatment. Desirable effects of treatment include, but are not limited to, preventing occurrence or reoccurrence of the disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.

[0049] The term "prevention" includes inhibition of the occurrence or development of a disease or condition or symptoms of a particular disease or condition. In some embodiments, a subject with a family history of cancer is a candidate for a prophylactic regimen. Generally, in the context of cancer, the term "prevention" refers to administration of a drug prior to the onset of signs or symptoms of cancer, particularly in a subject at risk for cancer.

[0050] The term "effective amount" refers to the amount or dose of a modified cell (e.g., red blood cell) or composition of the application, which elicits the desired effect in a patient in need of treatment or prevention, either as a single dose or as repeated doses. An effective amount can be readily determined by the attending diagnostician, as one skilled in the art, by the consideration of numerous factors, such as species of mammal; body mass, age and general health condition; the particular disease involved; extent or severity of disease; response of the individual patient; particular antibody being administered; mode of administration; bioavailability characteristics of the formulation administered; chosen dosing regimen; and use of any concomitant therapy.

[0051] The term "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount of the modified cells (e.g., red blood cells) or compositions of the application can vary according to factors such as the disease state, age, sex, and weight of the individual. A "therapeutically effective amount" preferably inhibits measurable parameters (e.g., uric acid levels, tumor growth rate, tumor volume, etc.) by at least about 20%, more preferably by at least about 40%, even more preferably by at least about 50%, 60%, or 70%, and still more preferably by at least about 80% or 90%, relative to an untreated subject.

[0052] The term "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Generally, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.

[0053] The term "pharmaceutical composition" refers to a composition that is in a form that allows the biologically active ingredient contained therein to be effective, and that does not contain additional ingredients that are unacceptable with respect to toxicity to the subject to which the composition will be administered.

[0054] II. Red Blood Cells (RBCs)

[0055] In humans, red blood cells are the most abundant cells in the circulatory system. Unlike other blood cells, red blood cells lack a nucleus and are flexible, allowing them to change shape to fit into blood vessels in the body, and primarily serve the function of oxygen supply in the body. Key protein markers on the surface of red blood cells allow them to circulate in the body for long periods of time without being cleared by macrophages, and thus have a long half-life, which makes them excellent candidates for drug carriers. Mature red blood cells, which lack a nucleus, do not contain any genetic material, and thus have good safety compared to other genetic and cell therapies.

[0056] Unless otherwise noted or otherwise apparent from context, references to red blood cells in the present disclosure generally refer to mature red blood cells. In certain embodiments, the RBCs are human RBCs, e.g., human native RBCs.

[0057] In some embodiments, the RBCs are not genetically engineered. In some embodiments, the present application provides red blood cells having an active agent conjugated thereto via a sortase-mediated reaction. In some embodiments, the active agent comprises a binding agent, a therapeutic agent, or a detection agent, including, for example, a protein, an antibody or a functional antibody fragment thereof, an antigen such as a tumor antigen, an MHC-peptide complex, a drug such as a small molecule drug (e.g., an anti-tumor agent, e.g., a chemotherapeutic agent), an enzyme (e.g., a functional metabolic enzyme such as UOX, or a therapeutic enzyme), a hormone, a cytokine, a growth factor, an antimicrobial agent, a probe, a ligand, a receptor, an immune tolerance inducing peptide, a targeting moiety, a prodrug, or any combination thereof. In some embodiments, the active agent is an anti-PDl antibody. In some embodiments, the active agent can be linked to the sortase receptor motif via a sortase recognition motif. In some specific embodiments, the active agent is a peptide molecule. In some embodiments, a membrane protein on the cell surface of the cell (e.g., red blood cell) is modified to conjugate a sortase recognition motif. In other embodiments, a membrane protein on the cell surface of the cell (e.g., red blood cell) is modified to conjugate a sortase receptor motif.

[0058] A "sortase receptor motif" refers to a motif that comprises an oligoglycine that serves as an amine nucleophile to attack the intermediate formed by the sortase and the recognition motif, and ultimately covalently couple to the recognition motif. In some embodiments, the sortase receptor motif is located at the N-terminus or C-terminus of a polypeptide. In some embodiments, the receptor motif comprises Gly-[Gly]n- (SEQ ID NO: 7), where n = 0-5.

[0059] A "sortase recognition motif" refers to a polypeptide that forms a thioester bond with a sortase molecule upon cleavage by the sortase molecule. In some embodiments, the sortase recognition motif comprises LPXTG, where X is any amino acid. In some embodiments, the sortase cleavage occurs between T and G.

[0060] In some embodiments, the present application contemplates the use of autologous cells isolated from an individual, e.g., autologous red blood cells, which are modified in vitro and then administered to the individual from which the autologous cells were isolated. In some embodiments, the present application contemplates the use of immunocompatible cells, e.g., red blood cells, which are of the same blood type (e.g., at least with respect to the ABO blood group system, and in some embodiments, the D blood group system) as the individual to whom the cells will be administered or can be a compatible blood type.

[0061] In some embodiments, the present application provides a method of improving the drug loading capacity of a cell (e.g., a red blood cell) by employing improved sortase variants, comprising contacting the cell (e.g., a red blood cell) with an active agent and allowing a conjugation reaction to occur in the presence of the improved sortase of the present application, thereby improving the drug loading capacity of the cell (e.g., a red blood cell).

[0062] In some embodiments, the present application provides cells (e.g., red blood cells) carrying higher drug loading, by employing the improved sortase of the present application, such that the cells (e.g., red blood cells) are contacted with an active agent and undergo conjugation reaction, thereby obtaining cells (e.g., red blood cells) carrying higher drug loading. In some embodiments, the cells (e.g., red blood cells) comprise a sortase receptor motif on the surface of their cell membrane, and the active molecule comprises a sortase recognition motif. In some embodiments, the active molecule is an anti-PD1 antibody. In some embodiments, the anti-PD1 antibody comprises a sortase recognition motif, and is linked to the RBC via a linker comprising a sortase receptor motif on the surface of the cell (e.g., red blood cell) membrane. In some embodiments, the cells (e.g., red blood cells) are modified such that their membrane proteins are covalently linked to the linker.

[0063] III. Sortase

[0064] Sortase refers to an enzyme that catalyzes a transpeptidation reaction by which a first molecule is conjugated to a second molecule via a transamidation. A molecule comprising a sortase recognition motif is referred to herein as a “sortase substrate”. In the context of the present application, the term sortase includes full-length sortases (e.g., full-length naturally occurring sortases), active sortase fragments, and modified sortase variants.

[0065] Sortases of the present application encompass all known various types of sortases, such as sortase A, sortase B, sortase C, and sortase D. In some embodiments, the sortase is a sortase A. The term “sortase A” refers to a class A sortase from any bacterial species or strain, often referred to as SrtA. In some embodiments, the sortase A is Staphylococcus aureus sortase A. In other embodiments, the sortase A is sortase A of S. pyogenes.

[0066] The motif that is typically recognized by sortase A is the short peptide LPXTG (SEQ ID NO: 8), where X is a standard or non-standard amino acid (e.g., X is selected from D, E, A, N, Q, K, or R). In some embodiments, the motif recognized by sortase A is LPETG (SEQ ID NO: 15). In some embodiments, the sortase recognition motif can be modified to improve its recognition efficiency, preferably, the LPETG is modified to improve its affinity to sortase, such as adding a G at the C-terminus of the recognition sequence, such as the modified sequence LPETGG (SEQ ID NO: 16). In some embodiments, the recognition motif is modified with Abz and DNP.

[0067] In addition, Sortase A can also recognize other motifs, for example, a motif in which the 4th position of the Sortase A recognition motif contains an "A", "S", "L" or "V" instead of "T". In some embodiments, the 5th position of the motif contains an "A" instead of "G". In some embodiments, the 2nd position of the motif contains a "G" or "A" instead of "P". In some embodiments, the 1st position of the motif contains an "I" or "M" instead of "L". Various recognition motifs for Sortase A are described in Pishesha et al. 2018.

[0068] Sortase A can achieve covalent conjugation of two entities in vitro by recognizing a substrate comprising a recognition motif. Specifically, Sortase A specifically recognizes a substrate comprising the motif LPXTG and cleaves between the threonine and glycine residues of the motif, forming a substrate thioester acyl-enzyme intermediate, which is then attacked by an amine nucleophile from a receptor motif comprising an oligoglycine, causing the decomposition of the intermediate, such that the entity comprising the receptor motif is covalently linked to the substrate comprising the recognition motif, and the Sortase A is regenerated. Motifs that can undergo an amine nucleophile attack reaction with a Sortase recognition motif are also referred to herein as Sortase receptor motifs, which typically comprise an oligoglycine sequence.

[0069] The specific catalytic activity of Sortases has been used for various protein engineering and bioconjugation purposes. With Sortase A, it is possible to introduce natural and synthetic functional entities into entities tagged with an LPXTG sequence, such as polymers (e.g. PEG), fluorophores, vitamins (e.g. biotin and folate), lipids, carbohydrates, nucleic acids, synthetic peptides and proteins (e.g. GFP), etc. In addition, Hangzhou Biomedicine Technology Co., Ltd. has successfully conjugated a variety of active substances to modified or unmodified red blood cells by engineering the LPXTG motif into the active substances and taking advantage of the properties of Sortases, thereby modifying red blood cells into advantageous drug carriers to achieve the corresponding therapeutic functions.

[0070] IV. Sortase Mutants

[0071] In some embodiments, the present application contemplates the use of naturally occurring variants of Sortases. The prior art discloses a wealth of structural information on Sortase A, including NMR or crystal structures of SrtA alone or in combination with a Sortase recognition sequence (see, e.g., Zong Y et al. J. Biol Chem. 2004, 279, 31383-31389). Sortase fragments having Sortase transamidation activity can be produced by recombinant techniques. For example, the fragment lacks the N-terminal transmembrane region required for non-catalytic activity, e.g., 25-60 amino acids from the N-terminus of the Sortase.

[0072] In some embodiments, the sequence of full-length wild-type S. aureus SrtA is set forth in SEQ ID NO: 1 (full length, GenBank Accession No: CAA3829591.1): 1 MKKWTNRLMT IAGVVLILVA AYLFSKPHID NYLHDKDKDE KIEQYDKNVK 51 EQASKDKKQQ AKPQIPKDKS KVAGYIEIPD ADIKEPVYPG PATPEQLNRG 101 VSFAEENESL DDQNISIAGH TFIDRPNYQF TNLKAAKKGS MVYFKVGNET 151 RKYKMTSIRD VKPTDVGVLD EQKGKDKQLT LITCDDYNEK TGVWEKRKIF 201 VATEVK

[0073] In some embodiments, the functional variant of S. aureus sortase A useful in the present application can be a S. aureus sortase A variant. In some embodiments, the sortase A mutant is a soluble truncated sortase A lacking the transmembrane region, e.g., lacking the N-terminal 25-60 (e.g., 30, 35, 40, 45, 50, 55, 59, or 60) amino acids. In some embodiments, the sortase A mutant comprises a fragment of about position 60 to about position 206 of wild-type S. aureus SrtA (numbering according to the sequence set forth in SEQ ID NO: 1).

[0074] On the basis of the truncated sortase A, further mutants were obtained by further modification of the corresponding fragment, e.g., the mutant enzyme having the following sequence:

[0075] In the present application, the amino acid mutation positions are determined by alignment with SEQ ID NO: 1. Methods for determining the amino acid positions corresponding to the mutation positions described herein are well known in the art. The corresponding amino acid residues in another polypeptide can be determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453) as implemented in the Needle program of the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277, preferably version 3.0.0 or later), with default parameters. Based on the well-known computer programs described above, the amino acid positions of the polypeptides of interest described herein can be routinely determined by one of skill in the art.

[0076] Accordingly, the present application provides a sortase A mutant comprising an amino acid sequence homologous to the amino acid sequence of wild-type sortase A (SEQ ID NO: 1) or a truncated mutant thereof (SEQ ID NO: 2). In some embodiments, the sortase mutant can further comprise one or more additional mutations. For example, the sortase mutant has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to SEQ ID NO: 1, or the sortase mutant has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to SEQ ID NO: 2. In some embodiments, the mutant further comprises one or more mutations selected from P94R, D124G, M155V, D160N, K162P, Y187L, E189R, K190E, and F200L (numbered according to the sequence shown in SEQ ID NO: 1) relative to SEQ ID NO: 1.

[0077] In some embodiments, the mutants of the present application have enhanced sortase A thermal stability and / or catalytic activity relative to the parent.

[0078] In some embodiments, the sortase A mutant has at least 50%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, at least 1000%, or even more increased thermal stability relative to the parent (e.g., SEQ ID NO: 2).

[0079] In some embodiments, the sortase A mutant has at least 50%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 500%, at least 600%, at least 700%, or even more increased maximum rate of activation (Vmax) relative to the parent (e.g., SEQ ID NO: 2).

[0080] In some embodiments, the sortase mutants obtained by the present application can further comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 conservative amino acid mutations that do not significantly affect the function / activity of the enzyme. Conservative amino acid mutations that do not significantly affect the activity of a protein are well known in the art.

[0081] V. Sortase Activity Assay

[0082] Methods of assaying sortase activity are known in the art.

[0083] Sortase reactions can be performed in any convenient vessel (e.g., microfuge tubes, microtiter plates, glass slides, silicon chips, filters, multi-channel chips, etc., any solid or semi-solid support). Reactions can be performed at any convenient temperature at which sortase reactions can be performed, e.g., sortase reactions are performed at temperatures between about 15°C and 50°C, preferably sortase reactions are performed at temperatures between 30°C and 40°C, more preferably sortase reactions are performed at temperatures between about 23°C and 37°C. In some embodiments, sortase reactions are performed at temperatures of about 37°C. In other embodiments, sortase reactions are performed at room temperature (i.e., about 20°C to 25°C) or at elevated temperatures (e.g., 40°C).

[0084] In sortase reactions, the molar ratio of enzyme to substrate containing a recognition motif is, e.g., between 1 : 100, e.g., between about 1 : 10, between 1 :5, between 1 :4, between 1 :3, between 1 :2. In other words, in some embodiments, the concentration of substrate containing a recognition motif in the reaction mixture is about 10 μΜ to about 10 mM, e.g., about 100 μΜ to about 1 mM, about 100 μΜ to about 5 mM, about 200 μΜ to about 1 mM, about 200 μΜ to about 800 μΜ, about 400 μΜ to about 600 μΜ.

[0085] In some embodiments, the concentration of substrate containing a sortase acceptor sequence is between about 1 μΜ and about 500 μΜ, e.g., about 15 μΜ to about 150 μΜ, about 25 μΜ to about 100 μΜ, about 40 μΜ to about 60 μΜ.

[0086] In some embodiments, the concentration of sortase in the reaction is about 1 μΜ to about 500 μΜ, e.g., about 15 μΜ to about 150 μΜ, about 25 μΜ to about 100 μΜ, about 40 μΜ to about 60 μΜ.

[0087] In some embodiments, the reaction mixture comprises a buffer. Those of skill in the art are well aware of a variety of buffers that can be used in the methods reported herein. In some embodiments, the reaction is performed at a pH between 6 and 8.5, e.g., at a pH between 6 and 8, at a pH between 6 and 7.5, at a pH between 6.5 and 8.5, at a pH between 7 and 8.5, at a pH between 7.5 and 8.5, at a pH between 7.0 and 8.5. In some embodiments, the reaction is performed at a pH between 7.3 and 7.8. Examples

[0088] The following examples further illustrate the application, however, it is understood that the examples are described by way of illustration and not by way of limitation and that modifications can be made by those skilled in the art.

[0089] The practice of the present application will employ, unless otherwise indicated, conventional chemistry, biochemistry, organic chemistry, molecular biology, microbiology, recombinant DNA techniques, genetics, immunology, and cell biology methods within the skill of the art. Experimental materials used are commercially available, unless otherwise stated. Techniques and conditions not specifically described in the examples were performed according to techniques and conditions described in the literature or according to the manufacturer's instructions. Reagents or instruments not specifically indicated were conventional products available through regular channels.

[0090] Example 1. Experimental materials

[0091] 1.1 Mutants of the present application

[0092] The truncated sortase SEQ ID NO: 2 was further mutated to obtain the mutants shown in Table 1. To facilitate purification, a 6xHis tag was added at the N-terminus of each mutant sequence.

[0093] Table 1. Mutants obtained in the experiment

[0094] The amino acid sequences of each mutant are as follows:

[0095] SrtA-V1:

[0096] SrtA-V2

[0097] SrtA-V3

[0098] SrtA-V4

[0099] 1.2 Culture medium

[0100] (1) Seed culture medium: 10 g / L peptone, 5 g / L yeast powder, 10 g / L NaCl;

[0101] (2) High-density fermentation medium 1: 5 g / L KH2PO4, 10 g / L K2HPO4·3H2O, 5 g / L (NH4)2SO4, 1 g / L anhydrous citric acid, 1 g / L MgSO4·7H2O, 10 g / L yeast powder, 50 g / L glycerol, 0.1 g / L vitamin B1, and 1 mL / L trace elements;

[0102] (6) Trace elements: 100 g L-1Fe(III) citrate, 18 g L -1 ZnCl3, 14.64 g L - 1 MnSO4.H2O, 0.75 g L -1 CuSO4.5H2O, 2 g L -1 Na2MoO4.2H2O, 2 g L -1 CaCl2.2H2O, 3.0 g L -1 H3BO3, 2.5 g L -1 CoCl2.6H2O, 2.5 g L-1NiSO4.6H2O and 100 mL HCl.

[0103] 1.3 Experimental reagents

[0104] Table 2. Drugs involved in this experiment

[0105] 1.4 Main solutions and reagents

[0106] Enzyme and substrate preparation method: Sortase A was diluted with PBS to 10 μΜ 1000 μl working solution; SrtA peptide was prepared with water to 1 mM stock solution, and NaOH was used to adjust the pH to 7-8; GL peptide was prepared with PBS to 4 mM stock solution, and NaOH was used to adjust the pH to 7-8.

[0107] Example 2. Construction, expression and purification of sortase mutants

[0108] 2.1 Strain construction

[0109] The nucleotide sequence encoding the sortase mutant (as shown in SEQ ID NO: 3-6) was constructed into the pET-28a(+) expression vector to obtain the plasmid pET-28a(+)-Sortase, which was then transformed into E. coli BL21(DE3) competent cells, and the LB plate was incubated at 37°C for 12 h. Then the colonies were picked, the nucleic acid of the colonies was extracted and PCR verification was performed. The positive transformants were picked and inoculated into test tubes containing LB medium and incubated at 37°C, 220 rpm. The plasmid was extracted and sequencing verification was performed. The strains with correct sequencing were inoculated and subcultured and the strains were stored, and the culture conditions were 37°C, 220 rpm.

[0110] 2.2 Seed liquid culture

[0111] The positive monoclonal strain obtained in Section 2.1 was subcultured for 8-10 h, and a clone was inoculated into a 250 mL flask containing 25 mL of LB medium, and cultured at 37°C, 220 rpm for 8-10 h.

[0112] 2.3 250mL flask culture

[0113] The secondary seed liquid obtained in Section 2.2 was inoculated into a 250 mL flask containing 50 mL of high-density fermentation medium at an inoculation amount of 2%, and cultured at 37°C, 220 rpm. After 2-3 h, IPTG was added, and the protein was induced at 30°C, 220 rpm.

[0114] 2.4 Cell disruption

[0115] After the induction of expression was completed, the bacterial cells in Section 2.3 were collected by centrifugation, and the wet bacterial cells were suspended with a cell lysis buffer at a volume ratio of 1:5. The cells were disrupted by using a high-pressure homogenizer, and the pressure was maintained at 700-900 psi, and multiple cycles were performed until the bacterial solution became clear. The solution was centrifuged at 14,000 g, 4°C for 30 min, and the supernatant was filtered through a 0.22 μm filter membrane and used.

[0116] 2.5 Protein purification

[0117] The supernatant obtained in Section 2.4 was sequentially subjected to Ni affinity chromatography, cation exchange chromatography purification, and UFDF according to the following conditions and steps, to obtain the corresponding sortase mutants (such as SEQ ID NO: 3-6).

[0118] 2.5.1 Buffer formula

[0119] (1) Ni affinity chromatography buffer:

[0120] Lysis solution: 50 mM Tris-HAC, + 200 mM NaCl + 5 mM imidazole (pH 7.5)

[0121] Washing solution: 50 mM Tris-HAC + 500 mM NaCl + 20 mM imidazole (pH 7.5)

[0122] Elution solution: 50 mM Tris-HAC + 50 mM NaCl + 250 mM imidazole (pH 7.5)

[0123] (2) Cation exchange chromatography buffer:

[0124] SPA buffer: 20 mM Tris-HAC, pH 7.5;

[0125] SPB buffer: 20mM Tris-HAC-1M NaCl, pH 7.5;

[0126] (3) UF / DF buffer:

[0127] PBS, pH 7.4.

[0128] 2.5.2 Experimental Procedure

[0129] 2.5.2.1 Ni affinity chromatography was used to capture the target protein.

[0130] (1) A gravity column filled with 2 mL of Ni Sepharose 6FF (Cytiva, 17531801) packing was equilibrated with 5 column volumes (CV) of lysis buffer at room temperature.

[0131] (2) Load the filtered supernatant obtained in Section 2.4 onto the affinity column and collect the flow-through liquid;

[0132] (3) Wash the affinity column with 20CV washing solution to remove unbound components and collect the washing solution;

[0133] (4) Elute the affinity column with 6CV elution buffer to harvest the target protein and collect the eluent;

[0134] (5) Take the loaded sample, flow-through solution, washing solution and elution solution respectively for SDS-PAGE electrophoresis detection;

[0135] 2.5.2.2 Cation exchange chromatography was used to purify the mutant protein.

[0136] Cation exchange chromatography utilizes Avant 25 (GE Healthcare) was operated primarily in accordance with the manufacturer's instructions.

[0137] (1) Equilibrate 5 mL of HiTrap SP FF (Cytiva, 17515701) chromatography column with SPA buffer until the conductivity and UV monitoring lines level out;

[0138] (2) The eluent obtained by Ni affinity chromatography was diluted with SPA buffer to a volume of 5 times and loaded at a flow rate of 2 ml / min.

[0139] (3) Treat the SP Sepharose FF column again with SPA buffer until the conductivity and UV monitoring lines level out;

[0140] (4) Use SPB buffer for linear gradient elution, set up a gradient of 2 column volumes to reach 20%, and then a gradient of 10 column volumes to reach 100%, while collecting the eluent at 4 ml / fraction.

[0141] (5) According to the peak map, select the appropriate fraction for SDS-PAGE detection;

[0142] (6) Based on the electrophoresis results, select the fraction with a purity of >90% in the main peak for combination, which is the target protein after purification.

[0143] 2.5.2.3 Ultrafiltration-diafiltration (UF / DF)

[0144] The target sample after cation exchange chromatography was concentrated and replaced into PBS using Amicon Ultra-15 10kDa (Merck) to make the target protein concentration reach 10mg / mL, and then 100μL / tube was aliquoted and stored frozen.

[0145] 2.5.3 Purification results

[0146] The results, as shown in Figures 1-4 and Table 3, show that through the above purification steps, the required high-purity sortase mutant is obtained.

[0147] Table 3. Yield of each step of Sortase mutant protein purification

[0148] Example 3. Detection of thermal stability of sortase mutants

[0149] The thermal stability of the mutants obtained by incubating the sortase mutants at 40°C for 2h was detected by transpeptidation reaction.

[0150] 3.1 Sortase A catalytic reaction

[0151] (1) The catalytic reaction system was set up according to Table 4:

[0152] Table 4. Reaction system

[0153] The fluorescent small peptide is synthesized by GenScript, with the sequence LPETGK containing Abz and DNP modification, i.e. Abz-LPETGK(Dnp). The substrate containing the acceptor motif is a nucleophilic attack small peptide synthesized by GenScript, with the sequence containing G oligopeptide, such as GGGSK (SEQ ID NO: 17).

[0154] (2) Abz-LPETGK(Dnp) was dissolved in ddH2O and adjusted to pH 6-7 with 0.5M NaOH to prepare a 4mM stock solution, then diluted with PBS (pH 7.4) by 2-fold gradient to form 7 concentrations for use, i.e. 4 / 2 / 1 / 0.5 / 0.25 / 0.125 / 0.0625mM. According to the reaction system in Table 4, 10μL of recognition motif was added to a non-transparent 96-well plate.

[0155] (3) The substrate nucleophile small peptide was dissolved in ddH2O and adjusted to pH 6-7 with 0.5M NaOH to prepare a 20mM stock solution, which was then diluted with PBS (pH 7.4) to 4mM for use. The mixture of nucleophile small peptide and enzyme was prepared according to the reaction system in Table 3, and then 90μL was added to the wells containing the recognition motif in the 96-well plate;

[0156] (4) The temperature of the enzyme reader was set to 30℃, and the pre-warm program was executed for 5min to make the reaction system temperature reach equilibrium;

[0157] (5) The enzyme was added to start the reaction, and the kinetic fluorescence monitoring program was executed, EX 320nm, EM 420nm, reading once every 23s, a total of 10min, and setting the shaking plate before the first reading and between each reading;

[0158] (6) The fluorescence enhancement rate and substrate concentration were fitted to the Michaelis equation, and the Km and Vmax values were obtained by nonlinear fitting in Origin 2018 to compare the relative catalytic efficiency.

[0159] In this series of catalytic reactions, His-Sortase (SEQ ID NO: 2) was used as a control to compare the catalytic activity of the mutants obtained in the present application.

[0160] 3.2 Experimental results

[0161] The maximum rate (Vmax) and appearance of each mutant before and after treatment are shown in Table 5, and the change rate of Vmax of each mutant before and after treatment is shown in Figure 5.

[0162] Table 5. Vmax and appearance of different mutants for end-terminal GGG-containing acceptor motif

[0163] The results show that the in vitro transpeptidation efficiency of the mutant obtained at room temperature is comparable to that of the control His-Sortase. After heating treatment, the maximum catalytic rate of the control His-Sortase enzyme is significantly reduced (by 88%), thus it is required to be purified and other operations at 2-8°C. The operation at 2-8°C makes the enzyme unable to be produced in large scale in practical application, i.e. the productivity is greatly reduced. After heating treatment, the maximum catalytic rate of the new mutant obtained in the application is basically unchanged, thus the purification can be performed at room temperature. These Sortase enzyme mutants which can be produced and purified at room temperature solve the industrial production problem, and can enlarge the production scale and reduce the cost. After heating treatment, the appearance of the control His-Sortase enzyme appears white turbidity, while the appearance of the mutant obtained in the application changes less. The above experimental results all show that the thermal stability of the control His-Sortase is poor, and the thermal stability of the four new mutants is obviously better than that of His-Sortase.

[0164] 3.3 DSC detection results of each mutant

[0165] Differential scanning calorimetry (DSC) is an analytical method for measuring the energy difference between a sample and a reference with temperature change under programmed temperature. With the increase of temperature, the change of protein structure is accompanied by the change of heat, which is recorded by the differential scanning calorimeter to the DSC curve. The starting temperature (Tonset) and the dissolution temperature (Tm) values of the protein are obtained by analyzing the DSC curve.

[0166] In the experiment, the DSC of the parent mutant His-Sortase and the newly obtained mutant was detected. The results are shown in Figures 6-7 and Table 6.

[0167] As shown in Figure 6, the starting temperature (Tonset) of His-Sortase in various buffer systems is less than 10°C. The smaller the Tonset value, the poorer the thermal stability. The results of Figure 7 show that the starting temperature (Tonset) of each mutant SrtA-V1, SrtA-V2, SrtA-V3 and SrtA-V4 obtained in the application is higher than 29°C, and the arrow in the figure shows the Tonset position of SrtA-V3 with the lowest starting temperature in each mutant.

[0168] Table 6. Starting temperature and Tm (Temperature maximum) values of the mutants

[0169] The results show that the thermal stability of the four newly screened mutants is increased to about 30°C, and the highest can be more than 33°C, compared with the parent sortase His-Sortase. This experimental result again shows that the thermal stability of the newly screened mutants is significantly improved compared with His-Sortase.

[0170] Example 4. Detection of the affinity of sortase mutants to single G

[0171] The general acceptor motif of sortase contains three G (GGG), and the sortase after mutation and selection can recognize an acceptor motif containing only one G (G). This embodiment detects the affinity of each mutant to a nucleophilic substrate with only one G at the N-terminus by a transpeptidation reaction.

[0172] 4.1 Sortase A catalytic reaction

[0173] (1) The catalytic reaction system is configured according to Table 7:

[0174] Table 7. Reaction system

[0175] The fluorescent small peptide is synthesized by GenScript, and the sequence is LPETGK containing Abz and DNP modification, i.e. Abz-LPETGK(Dnp). The substrate containing the acceptor motif (i.e. the nucleophilic attack small peptide) is synthesized by GenScript, and the sequence is an oligopeptide containing one G, such as G-FLAG (Biotin) peptide.

[0176] (2) Abz-LPETGK(Dnp) is dissolved in ddH2O and adjusted to pH 6-7 with 0.5M NaOH to configure a 4mM stock solution, and then diluted with PBS (pH 7.4) by 2-fold gradient to form 7 concentrations for use, i.e. 4 / 2 / 1 / 0.5 / 0.25 / 0.125 / 0.0625mM. According to the reaction system in Table 4, 10μL of the recognition motif is added to the non-transparent 96-well plate.

[0177] (3) The substrate nucleophilic attack small peptide is dissolved in ddH2O and adjusted to pH 6-7 with 0.5M NaOH to configure a 20mM stock solution, and then diluted with PBS (pH 7.4) to 4mM for use; according to the reaction system in Table 3, the mixture of nucleophilic attack small peptide and enzyme is configured, and then 90μL is added to the wells containing the recognition motif in the 96-well plate;

[0178] (4) The temperature of the microplate reader is set to 30°C, and the pre-warm program is executed for 5min to make the temperature of the reaction system reach equilibrium;

[0179] (5) Add enzyme to start the reaction, perform the kinetic fluorescence monitoring program, EX 320 nm, EM 420 nm, read every 23 s, a total of 10 min, set the vibration plate before the first reading and between each reading;

[0180] (6) Fit the fluorescence enhancement rate and substrate concentration to the Michaelis equation, and use the Michaelis equation in Origin 2018 for non-linear fitting to obtain the Km and Vmax values. Compare the relative catalytic efficiency.

[0181] In this series of catalytic reactions, the catalytic activity of the mutants obtained in the present application was compared with His-Sortase (SEQ ID NO: 2) as a control.

[0182] 4.2 Experimental results

[0183] The experimental results are shown in Table 8.

[0184] Table 8. Vmax, Km values of different mutants for small nucleophilic peptides containing only a single G at the N terminus

[0185] The results show that the newly screened mutants have comparable or even higher catalytic efficiency compared with the parent; among them, the Vmax value of SrtA-V3 increases by 1.96 times, and the Vmax value of SrtA-V4 increases by 2.33 times. The smaller the value of Km, the higher the catalytic efficiency of the enzyme. In Table 8, the numerical results of Km also show that the mutants have high affinity for G small peptides containing only one glycine; among them, SrtA-V3 is reduced to 56.4 μM. Considering the excellent thermal stability performance of the new mutants, it is shown that the newly screened mutants can efficiently catalyze the conjugation of drugs with proteins containing a single glycine on the surface of red blood cell membranes, thereby increasing the drug loading capacity of red blood cells, and are more preferred industrial sortases.

[0186] Example 5. Preparation of red blood cells with high drug loading capacity by sortase mutant catalysis

[0187] This example studies the improvement of the drug loading capacity of red blood cells containing a sortase receptor motif (containing GGG) by sortase mutants.

[0188] Red blood cells were isolated from peripheral blood of C57 / B6 mice (Shanghai Jihui Experimental Animal Breeding Co., Ltd.) using density gradient centrifugation. The isolated red blood cells were washed three times with PBS and then pretreated with 5 mM tris(2-carboxyethyl)phosphine (TCEP, Sigma) at 30°C for 1 hour. TCEP treatment reduced extracellular disulfide bonds in red blood cell membrane proteins, resulting in free sulfhydryl groups on the red blood cell membrane surface. The pretreated red blood cells were washed three times with PBS and then reacted with adapters containing receptor motifs G1 and G2 at room temperature for 30 minutes, allowing the adapters to attach to the red blood cell membrane proteins. G1 is GGGSK(6-Maleimidohexanoic Acid) (SEQ ID NO:9), wherein 6-Maleimidohexanoic Acid is conjugated to the ε-amino group of the lysine side chain; G2 is 6-Maleimidohexanoic Acid-K(GGG)GGGK(GGG) (SEQ ID NO:10), wherein KGGGK constitutes the main chain of G2, and 6-Maleimidohexanoic Acid is attached to the α-amino group of the N-terminal lysine; "(GGG)" indicates that a small peptide composed of three glycines is conjugated to the ε-amino group of the lysine side chain to form a branch chain. This small peptide contains two branches composed of GGG, which are conjugated to the ε-amino groups of the two lysine side chains, respectively.

[0189] Following conventional methods, the heavy chain of the recognition motif LPETG and the anti-PD1 antibody was constructed into a fusion protein containing the motif for use.

[0190] In the presence of the sorting enzyme mutant obtained in this application, 1×10 9 / mL of RBCs containing linkers were reacted with anti-PD1 antibody containing recognition motifs. In the conjugation reaction, the concentration of sorting enzyme mutant was 10 μM and the concentration of anti-PD1 antibody was 25 μM.

[0191] The amino acid sequence of the heavy chain LPETG of the anti-PD1 antibody is shown below:

[0192] The amino acid sequence of the light chain of the anti-PD1 antibody is shown below:

[0193] The amount of anti-PD1 antibody carried by erythrocytes under the catalysis of different sortase enzymes was detected by Fc ELISA.

[0194] Specifically, by the routine operation of ELISA enzyme-linked immunoassay, PD1 is coated on the ELISA detection plate, and the red blood cell membrane is lysed by RIPA to release the anti-PD1 antibody. The lysate is added to the ELISA detection plate coated with PD1 at a suitable dilution, incubated to allow the anti-PD1 antibody to bind to PD1, then a detection secondary antibody coupled with HRP is added to bind the Fc end of the anti-PD1 antibody, HRP reaction substrate (TMB) is added for color development reaction, the light absorption of each sample is detected at 450 nm wavelength, and compared with the light absorption of the standard, so as to obtain the drug loading of the anti-PD1 antibody coupled on the red blood cells.

[0195] The results are shown in Tables 9-10.

[0196] Table 9. Drug loading of red blood cells coupled with PD1 antibody catalyzed by different mutants * The amount of PD1 antibody carried per milliliter of red blood cell preparation; ** The number of red blood cells contained per milliliter; *** The amount of PD1 antibody carried per 1010 red blood cells; 10 The amount of PD1 antibody carried on red blood cells;

[0197] As can be seen from the results in Table 9, regardless of using the acceptor motif G1 or G2, the sortase mutants obtained by the present application do not significantly affect the drug loading capacity of red blood cells, and they still have a high enough drug loading capacity of red blood cells, indicating that the sortase mutants retain the enzymatic reaction ability of the parent. Among them, SrtA-V2 makes the drug loading capacity of red blood cells carrying acceptor motif G2 increase by 16% relative to the parent sortase. Since the thermal stability of the new mutant is significantly improved, its application value is much greater than that of the control from the perspective of industrial production needs.

[0198] Table 10. Drug loading of red blood cells of different species coupled with PD1 antibody catalyzed by different mutants * The amount of PD1 antibody carried per milliliter of red blood cell preparation; ** The number of red blood cells contained per milliliter; *** The amount of PD1 antibody carried per 1010 red blood cells;

[0199] The results in Table 10 show that for the drug loading capacity of human red blood cells, the catalytic activity of all new mutants has been improved to varying degrees (from 5% to 28%) compared with the control. In the experiment of model animal mouse, the catalytic activity of the other three new mutants also has different degrees of improvement (from 8% to 17%) relative to the control, except for SrtA-V1.

[0200] Example 6. Catalytic time course of sortase mutants under different small peptide substrates and in vitro transpeptidation comparison of different enzyme concentrations

[0201] The present example studies the time course of the mutant catalyzing transpeptidation reaction in vitro under different NH2-G small peptide substrates, and the efficiency of the mutant catalyzing transpeptidation in vitro under different enzyme concentrations, and the efficiency of the mutant catalyzing transpeptidation is reflected by the accumulation of the ligation product His6-EGFP-LPET-G-peptide.

[0202] Table 11. Mutant time course reaction system (100 μL)

[0203] For the study of catalytic time course, the system was configured according to Table 11, and the system was placed in 37°C reaction for different time (0-120 min), and the results are shown in Figure 8a. The accumulation of ligation product catalyzed by each mutant changed with the increase of reaction time. Mutants SrtA-V2 and SrtA-V3 accumulated more ligation product than the control His-Sortase.

[0204] Table 12. Mutant different enzyme concentration reaction system (100 μL)

[0205] For the catalytic efficiency of different enzyme concentrations, the system was configured according to Table 12. After the reaction system was placed in 37°C water bath for 2h, 10 μL of the reaction system was taken, 20 μL of 2X loading buffer was added to terminate the reaction, and 10 μL of it was taken for SDS-PAGE detection. The results are shown in Figure 8b. The accumulation of ligation product catalyzed by each mutant was positively correlated with the increase of enzyme concentration. Among them, mutants SrtA-V2 and SrtA-V3 had higher transpeptidation efficiency than the control His-Sortase. At the same time, they could obtain higher product accumulation under the condition of low enzyme concentration.

[0206] Example 7. Sortase mutant catalyzing PBMC cell coupling GFP protein

[0207] The present example studies the ability of sortase mutants to couple green fluorescent (GFP) protein to PBMC cells (including T cells, NK cells, macrophages, DC cells, B cells) containing sortase receptor motif (containing GGG).

[0208] PBMC cells were isolated from the peripheral blood of C57 / B6 mice (Shanghai Jihui Experimental Animal Breeding Co., Ltd.) by density gradient centrifugation. The isolated PBMC cells were washed with PBS for 3 times, and then the PBMC cells were pretreated with 5 mM tris(2-carboxyethyl)phosphine at 30°C for 1 hr. The disulfide bonds located outside the cell membrane protein of the PBMC cells were reduced by the treatment of TCEP, so that the membrane surface of the PBMC cells contained free thiol groups. The pretreated PBMC cells were washed with PBS for 3 times, and then reacted with the above treated PBMC cells at room temperature for 30 min by using a linker containing the receptor motif G1 (SEQ ID NO: 9), so that the corresponding linker was conjugated to the membrane protein of the PBMC cells, respectively.

[0209] According to the conventional method, the recognition motif LPETG was constructed into a fusion protein containing the motif with the GFP protein for use.

[0210] In the presence of the sortase mutants obtained in the present application, 1 x 10 7 PBMC cells containing linkers were reacted with GFP fusion proteins containing recognition motifs at a concentration of 1-10 μM. In the conjugation reaction, the concentration of the sortase mutant was 1-5 μM, and the concentration of the GFP protein was 1-10 μM.

[0211] The amino acid sequence of GFP-LPETG is as follows:

[0212] The labeling effect of GFP on the membrane of PBMC cells was monitored by flow cytometry method (Cytoflex, Beckman), and the positive proportion of conjugated GFP protein in T cells, NK cells, monocyte macrophages and B cells was analyzed by specific antibodies CD3, CD19, CD14, CD16 and CD56, thereby characterizing the ability of the sortase mutants of the present application to couple GFP to PBMC cells. The results are shown in Figure 13. It is shown that the sortase mutants of the present application can catalyze the successful coupling of GFP-LPETG to the cell membranes of T cells, NK cells, macrophages and B cells.

[0213] SEQUENCE LISTING

Claims

1. A sortase mutant having an amino acid sequence which is at least 90% identical to a parent sortase as set forth in SEQ ID NO: 2 and comprising one or more mutations selected from P94R, D124G, M155V, D160N, K162P, Y187L, E189R, K190E and F200L, wherein the amino acid positions are numbered according to SEQ ID NO:

1.

2. The sortase mutant of claim 1 having an amino acid sequence which is at least 90% identical to a parent sortase as set forth in SEQ ID NO: 2 and comprising the mutations D124G / M155V / Y187L / E189R, D124G / M155V / D160N / Y187L / E189R, P94R / D124G / M155V / Y187L / E189R or P94R / D124G / M155V / K162P / Y187L / E189R / F200L.

3. The sortase mutant of claim 1 or 2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO:

6.

4. A polynucleotide molecule encoding the sortase mutant of any one of claims 1 to 3.

5. A vector comprising the polynucleotide molecule of claim 4.

6. A cell comprising the polynucleotide molecule of claim 4 or the vector of claim 5.

7. A method of coupling an active molecule to a cell, comprising contacting the cell with the sortase mutant of any one of claims 1 to 3 under conditions suitable for the active molecule to be coupled to the cell by the sortase mutant.

8. The method of claim 7, wherein the cell is a peripheral blood mononuclear cell, such as a T cell, NK cell, macrophage, DC cell, B cell, or preferably a red blood cell.

9. The method of claim 7 or 8, wherein the cell membrane surface of the cell comprises a sortase receptor motif and the active molecule comprises a sortase recognition motif.

10. The method of claim 9, wherein the sortase recognition motif is, for example, the amino acid sequence LPXTG, LPETGK, LPETGGK.

11. Use of the sortase mutant of any one of claims 1 to 3 for the directed coupling of molecules.

12. Use of the sortase mutant of any one of claims 1 to 3 for the directed coupling of an active molecule to a cell membrane.

13. The use of claim 12, wherein the cell is a peripheral blood mononuclear cell, such as a T cell, NK cell, macrophage, DC cell, B cell, or preferably a red blood cell.

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