Biotin ligase mutant, coding gene thereof, expression vector, recombinant cell and application of biotin ligase mutant

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to a biotin ligase mutant as well as a coding gene, an expression vector, a recombinant cell and application thereof. The amino acid sequence of the biotin ligase mutant is as shown in SEQ ID NO. 2. The biotin ligase (Bar A) mutant provided by the invention can be efficiently expressed in eukaryotes, the expression level of the biotin ligase (Bar A) mutant is obviously improved in mammalian cells compared with a wild type, and the biotin ligase (Bar A) mutant has good catalytic activity after expression, can effectively catalyze biotinylation of protein containing a biotin receptor sequence, and has good application prospects. The method has a wide application prospect in the fields of biotinylated protein preparation, and protein labeling, positioning, immunodetection, affinity purification, function research and the like based on a biotin-avidin system (BAS).

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and in particular to a biotin ligase mutant, its encoding gene, expression vector and recombinant cell and applications thereof. Background Art

[0002] The strong binding between biotin and avidin with high affinity and the characteristics of multi-stage amplification effect make the biotin / avidin system (BAS) a signal amplification labeling technology and a widely used tool with high affinity, high sensitivity, strong specificity and good stability. It has shown extremely important value in protein labeling, localization, immunoassay, affinity purification and functional research. Conventional applications of BAS include: (1) protein labeling and tracking: by biotinylating the target protein and using avidin modified with a detection marker (such as quantum dots, fluorescein) to specifically bind to the biotin molecule to produce a color reaction, the target protein can be tracked and located in living cells; (2) immunoassay: by labeling the protein (or antibody) with biotin, the avidin modified with the detection marker is bound to or bridged with free biotin, and the trace amount of antigen / antibody can be qualitatively or quantitatively detected; (3) protein interaction research: by fusing the biotin ligase with the target protein, the adjacent protein is labeled with biotin through enzyme-catalyzed covalent modification, and finally the biotin-labeled protein and its interacting protein are enriched by avidin magnetic beads for mass spectrometry identification, which can analyze the interaction network between proteins or the information of adjacent proteins; (4) protein affinity purification: by labeling the ligand protein with biotin, mixing it with the receptor protein, and using a chromatography column fixed with avidin to capture the ligand-receptor complex to achieve one-step purification of the receptor protein, which is suitable for the preparation of recombinant proteins.

[0003] One of the key applications of the BAS system is biotin labeling of proteins. Currently, the most commonly used methods include in vitro chemical methods and in vivo enzymatic methods. Because the biotinylation sites of in vitro chemical methods are randomly labeled, steric hindrance caused by biotinylation at certain sites can affect protein activity, and in vivo labeling in living cells is difficult, thus significantly limiting their application. Labeling techniques that use enzymes to conjugate biotin to proteins are becoming increasingly widely used. Biotin ligases (BPL or Bir A) are a class of enzymes found in prokaryotes and eukaryotes that catalyze the biotinylation of proteins containing biotin acceptor sequences (such as BAT or BSP sequences). These enzymes, also known as biotin acetyl-CoA synthetases, catalyze the biotinylation of target proteins with extremely high specificity. For example, Escherichia coli BirA can activate biotin in the presence of ATP to form a biotin-AMP intermediate, which then covalently binds the activated biotin to a lysine (K) residue in the protein's biotin acceptor sequence. Therefore, by fusion-expressing the biotin receptor sequence with the recombinant target protein, the expressed product can be biotin-labeled in vivo or in vitro via the catalytic action of the Bir A enzyme. Bir A enzymatic labeling offers clear modification sites, uniform labeling, and excellent reproducibility, while also minimizing the protein's native activity. This method represents a promising approach for in vivo protein activity labeling.

[0004] Most of the biotin ligases currently used come from prokaryotes, with Bir A (UniProtID: P06709) from Escherichia coli being the most widely used. However, the expression activity of these enzymes in eukaryotic cells is limited, and the efficiency of in vivo biotin labeling when co-expressed with the target protein still needs to be improved. Summary of the Invention

[0005] To address the problems of low expression efficiency and / or low enzyme activity of biotin ligase in eukaryotic cells in the prior art, the present invention provides a novel biotin ligase mutant and its encoding gene, as well as an expression vector and recombinant cells containing the encoding gene. Furthermore, the present invention provides the use of the biotin ligase mutant, its encoding gene, expression vector or recombinant cell in the preparation of biotinylated proteins.

[0006] The present invention is specifically achieved through the following technical solutions:

[0007] The first aspect of the present invention provides a biotin ligase mutant, the amino acid sequence of which is shown in SEQ ID NO.2.

[0008] The second aspect of the present invention provides a gene sequence encoding the biotin ligase mutant described above.

[0009] Furthermore, the gene sequence encoding the biotin ligase mutant is shown in SEQ ID NO.3.

[0010] The third aspect of the present invention provides a biotin ligase expression vector, which includes the gene sequence encoding the biotin ligase mutant as described above.

[0011] Optionally, the expression vector further comprises a gene sequence encoding a promoter, an enhancer, a terminator, a purification tag and / or a screening tag.

[0012] Furthermore, the expression vector is pcDNA3.4.

[0013] In a fourth aspect, the present invention provides a recombinant cell comprising the gene sequence encoding the biotin ligase mutant as described above or the biotin ligase expression vector as described above.

[0014] Furthermore, the recombinant cells are 293F cells.

[0015] In a fifth aspect, the present invention provides use of the biotin ligase mutant or its encoding gene as described above, or the biotin ligase expression vector as described above, or the recombinant cell as described above in preparing a biotinylated protein.

[0016] Furthermore, the preparation of the biotinylated protein includes in vivo or in vitro.

[0017] Furthermore, the preparation of the biotinylated protein in vitro comprises the following steps: co-incubating the biotin ligase mutant, the target protein to be labeled, and biotin under appropriate conditions.

[0018] Furthermore, preparing the biotinylated protein in vivo comprises the following steps:

[0019] S1. Connect the Avi Tag gene to the upstream or downstream of the target protein gene, insert the fusion gene into the expression vector, and construct the target protein expression vector;

[0020] S2. Transfecting the target protein expression vector and the biotin ligase expression vector into 293F cells to obtain recombinant cells that co-express the target protein and biotin ligase;

[0021] S3. Cultivate the recombinant cells and obtain the biotin-labeled target protein through purification.

[0022] Furthermore, step S2 includes: transfecting a biotin ligase expression vector into 293F cells, obtaining a 293F cell line stably expressing biotin ligase through screening, and transfecting a target protein expression vector into a 293F cell line stably expressing biotin ligase to obtain recombinant cells co-expressing the target protein and biotin ligase.

[0023] The advantages and positive effects of the present invention are:

[0024] The biotin ligase (Bir A) mutant provided by the present invention can be efficiently expressed in eukaryotes. Compared with the wild type, its expression level in mammalian cells is significantly improved. After expression, it has good catalytic activity and can effectively catalyze the biotinylation of proteins containing a biotin receptor sequence. With an antibody as the target protein to be biotinylated, the antibody gene fused with the Avi Tag is co-expressed in stably transfected cells expressing the Bir A mutant. The Bir A mutant can effectively catalyze the biotinylation of the antibody, significantly improving the biotinylation efficiency of the antibody. The labeling efficiency of the enzymatic biotinylation in vivo can reach an efficiency comparable to that of in vitro enzymatic labeling, and is basically equivalent to the currently commonly used in vitro chemical labeling method. It has broad application prospects in the fields of preparing biotinylated proteins and protein labeling, localization, immunoassay, affinity purification, and functional research based on the biotin-avidin system (BAS). BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0026] Figure 1 is a map of the biotin ligase expression vector of an embodiment of the present invention;

[0027] Figure 2 This is a fluorescence microscopy image of 293F cells expressing a biotin ligase expression vector according to an embodiment of the present invention;

[0028] Figure 3 This is a diagram showing the results of immunoblotting of 293F cell lysates expressing a biotin ligase expression vector in an embodiment of the present invention;

[0029] Figure 4 Figure 1 is a map of the antibody expression vectors of the embodiments of the present invention, wherein from left to right are the antibody light chain and heavy chain expression vectors pCDNA3.4;

[0030] Figure 5This is an electrophoretic diagram of the purified antibody in 293F cells co-expressing a biotin ligase mutant and an antibody expression vector according to an embodiment of the present invention;

[0031] Figure 6 This is a graph showing the results of detecting antibody biotinylation in 293F cells co-expressing a biotin ligase mutant and an antibody expression vector according to an embodiment of the present invention;

[0032] Figure 7 This is a graph showing the results of detecting the biotinylation efficiency of antibodies in 293F cells co-expressing a biotin ligase mutant and an antibody expression vector according to an embodiment of the present invention. DETAILED DESCRIPTION

[0033] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to the examples. Unless otherwise specified, the equipment and reagents used in each example and test example can be obtained from commercial sources. The specific examples described herein are only used to illustrate the present invention and are not intended to limit the present invention.

[0034] Based on the information contained in this application, it will be readily apparent to those skilled in the art that various changes can be made to the precise description of the present invention without departing from the spirit and scope of the appended claims. It should be understood that the scope of the present invention is not limited to the defined processes, properties, or components, as these embodiments and other descriptions are provided merely to illustrate specific aspects of the present invention. In fact, various changes that a person skilled in the art or related fields would clearly be able to make to the embodiments of the present invention are encompassed within the scope of the appended claims.

[0035] For a better understanding of the present invention and not to limit the scope of the present invention, all numbers used in this application to express amounts, percentages, and other numerical values ​​should be understood as modified by the word "about" in all cases. Therefore, unless otherwise specified, the numerical parameters listed in the specification and the appended claims are approximate values, which may vary depending on the desired properties to be obtained. Each numerical parameter should at least be regarded as obtained based on the reported significant figures and by conventional rounding methods.

[0036] In addition, it should be noted that, unless otherwise defined, in the context of the present invention, the scientific and technical terms used should have the meanings commonly understood by those skilled in the art.

[0037] The terms "include", "comprising", "containing", "having" and the like are non-limiting in meaning, that is, other steps and other components that do not affect the results may be added.

[0038] The term "and / or" should be regarded as a specific disclosure of each of the two specified features or components with or without the other. For example, "A and / or B" will be regarded as including the following situations: (i) A, (ii) B, and (iii) A and B.

[0039] The term "gene" refers to the complete nucleotide sequence required to produce a polypeptide chain or functional RNA. A gene may comprise several operably linked nucleic acid segments, such as a 5' noncoding region (UNTR), a coding sequence, and a 3' noncoding region (UNTR) containing a polyadenylation site. The NTR generally regulates gene expression, such as promoters and terminators.

[0040] The term "fusion gene" has the same meaning and refers to any gene that is not a native gene and includes regulatory sequences and coding sequences that are not found together in nature. Therefore, a fusion gene may include regulatory sequences and coding sequences derived from different organisms, or regulatory sequences and coding sequences derived from the same organism but arranged in a manner different from that found in nature, such as the fusion sequence of the antibody heavy chain of the present invention and the Avi Tag.

[0041] The term "vector" refers to a self-replicating DNA molecule that transfers the target gene into the host cell, and is often in the form of a circular double-stranded DNA molecule. A vector containing an exogenous gene is a recombinant vector.

[0042] The term "expression vector" allows the expression of a target gene inserted into the vector in a host cell and contains regulatory elements, such as a promoter and / or terminator, for expression in a specific host cell. The expression vector is introduced into an appropriate host cell to enable the expression of the inserted target gene.

[0043] The terms "introduction" or "transfer" refer to the transfer of a gene of interest into a host cell, resulting in genetically stable inheritance. The introduced nucleic acid molecule may be in the form of a plasmid that is retained in the host cell, or may be integrated into the host cell genome. Nucleic acid molecules and / or vectors may be introduced into a host cell by methods such as "transfection," "transformation," or "transduction." Host cells containing the introduced nucleic acid molecule are referred to as "transgenic," "recombinant," "transformed," or "engineered" organisms. Vector introduction into host cells may be performed using conventional techniques well known to those skilled in the art.

[0044] Unless otherwise indicated, the terms "nucleotide," "nucleic acid," "nucleic acid molecule," and "nucleic acid fragment" are used interchangeably in the context of the present invention. The terms "gene," "nucleic acid sequence," "nucleotide sequence," or "nucleic acid molecule" as used herein refer to polymeric forms of nucleotides of any length, either ribonucleotides or deoxyribonucleotides, and the terms refer only to the primary structure of the molecule.

[0045] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, specific embodiments of the present invention are described in detail below.

[0046] The Escherichia coli biotin ligase BirA catalyzes the biotinylation of proteins containing a biotin ligase recognition tag with high specificity. It recognizes the biotin carboxyl carrier protein (BCCP), a subunit of acetyl-CoA carboxylase, or proteins containing a biotin acceptor sequence, and specifically labels the biotin on lysine residues. The most commonly used peptide sequence for BirA catalytic biotinylation is a 15-amino acid peptide, the Avi Tag, with the protein sequence GLNDIFEAQKIEWHE. The Avi Tag possesses a biotinylated lysine residue that can be covalently attached to a biotin molecule by the biotin ligase (BirA). This reaction occurs efficiently and specifically both in vitro and in vivo. Therefore, proteins can be fused with the BCCP or biotin acceptor sequence and specifically biotinylated using BirA in vivo (during expression) or in vitro (after purification).

[0047] Bir A has important applications in catalyzing specific biotin labeling in vitro and in vivo. However, the Escherichia coli biotin ligase BirA has low expression efficiency and enzymatic activity in eukaryotic cells, resulting in inefficient biotinylation of target proteins. This limits its widespread application in eukaryotic expression systems, particularly in eukaryotic protein-protein interaction studies and functional protein detection.

[0048] Based on this, the present invention provides a biotin ligase (Bir A) mutant, which has amino acid substitutions (F271S / Q295L / A319V) at three positions 271, 295, and 319 compared to wild-type Bir A. The amino acid at position 271 is mutated from F to S (F271S), the amino acid at position 295 is mutated from Q to L (Q295L), and the amino acid at position 319 is mutated from A to V (A319V); its amino acid sequence is shown below:

[0049] MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRDWGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSGDACIAEYQQAGRGRRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRK LGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGIMAMRRVEESVVNQGWITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNSINRPVKLIIGDKEIFGISRGIDKLGALLLEQDGIIKPWMGGEISLRSVEK (see SEQ ID NO.2), the mutation site is shown in bold shade.

[0050] The present invention modifies the C-terminus of the wild-type Escherichia coli biotin ligase Bir A. The resulting biotin ligase mutant is more consistent with the expression pattern of eukaryotes and exhibits good catalytic activity after expression in eukaryotic cells. It can effectively catalyze the biotinylation of proteins containing a biotin receptor sequence. Using a mammalian expression system as an example to express the Bir A mutant gene, tests have found that under the same culture system and conditions, the Bir A mutant can enhance expression in mammalian cells, with its expression level significantly higher than that of wild-type Bir A. Furthermore, using an antibody as the target protein to be biotinylated, and co-expressing an antibody gene expression vector fused to an Avi Tag in stably transfected cells expressing the Bir A mutant, the Bir A mutant can effectively biotinylate the endogenous target protein, significantly improving the antibody biotinylation efficiency. The in vivo enzymatic biotinylation labeling efficiency can reach an efficiency comparable to that of in vitro enzymatic labeling, and is essentially equivalent to the commonly used in vitro chemical labeling method. It has broad application prospects in the preparation of biotinylated proteins and protein labeling, localization, immunoassay, affinity purification, and functional research based on the biotin-avidin system (BAS).

[0051] Another embodiment of the present invention provides a gene sequence encoding the biotin ligase mutant (F271S / Q295L / A319V) as described above.

[0052] The advantages of the gene sequence encoding the biotin ligase mutant over the prior art are the same as those of the biotin ligase mutant described above, and will not be repeated here.

[0053] Genes include DNA molecules (e.g., genomic DNA or cDNA) and / or RNA molecules (e.g., mRNA), which can be single-stranded or double-stranded. The gene sequence can be derived from the amino acid (AA) sequence of the biotin ligase mutant using conventional methods, such as codon coding rules. The full-length sequence or fragments thereof can generally be obtained by PCR amplification or synthetic methods.

[0054] Illustratively, the gene (DNA) sequence encoding the biotin ligase mutant described above is shown in SEQ ID NO.3.

[0055] Those skilled in the art will appreciate that, due to the degeneracy of the genetic code, gene sequences other than those exemplified above can also encode the biotin ligase mutant of the present invention. Therefore, the sequences exemplified above should not be used as a limitation on the scope of protection of the present invention.

[0056] Another embodiment of the present invention provides a biotin ligase expression vector, which includes the gene sequence described above.

[0057] Optionally, the biotin ligase expression vector also includes gene sequences encoding expression elements such as a promoter and / or terminator. The promoter is located upstream of the biotin ligase mutant, and the terminator is located downstream of the biotin ligase mutant. That is, when all the aforementioned expression elements are included, the gene linkage order from upstream to downstream is: promoter - biotin ligase mutant - terminator. The promoter initiates transcription of Bir A, and the terminator terminates transcription of Bir A, forming a complete biotin ligase expression cassette. The promoter and terminator are selected based on the type of expression vector and host cell. When expressing Bir A, the expression elements can also be selected based on actual needs. For example, if the selected vector already contains a promoter or terminator, there is no need to add these expression elements upstream and downstream of the biotin ligase mutant gene.

[0058] Optionally, the biotin ligase expression vector further comprises a gene sequence encoding an enhancer, which is used to enhance the transcriptional activity of the promoter.

[0059] Optionally, the biotin ligase expression vector further includes a gene sequence encoding a purification tag. The purification tag is used to facilitate the separation and purification of Bir A and can be located upstream or downstream of the biotin ligase mutant according to actual needs. The purification tag can be selected from one or more of 6×His, 8×His, Trx, 3×FLAG, GST, strep(II), HA, GFP, cMyc, and mFC. Those skilled in the art can specifically set the tag type according to the purification requirements to adapt to different purification systems and equipment, and the present invention is not limited to this.

[0060] Optionally, the biotin ligase expression vector further comprises a gene sequence encoding a screening tag. The screening tag is used to facilitate the screening of positive recombinant cells transformed with the expression vector, and includes but is not limited to an antibiotic encoding gene or a fluorescent protein encoding gene, such as a puromycin encoding gene or a red fluorescent protein encoding gene.

[0061] Optionally, the expression vector mentioned above includes a prokaryotic expression vector, a eukaryotic expression vector or a viral expression vector (such as a lentivirus or adenovirus). Correspondingly, the host cells transformed or transfected by the expression vector can be prokaryotic cells and eukaryotic cells, which are selected according to the type of expression vector. For example, when it is a prokaryotic expression vector, the host cell is selected as a prokaryotic cell. Examples of commonly used prokaryotic cells include Escherichia coli (such as DH5α, JM109, BL21, W3110), Bacillus (such as Bacillus subtilis, Bacillus thuringiensis), Corynebacterium, Pseudomonas, Zymomonas mobilis, Streptomyces, etc.; when it is a eukaryotic expression vector, the host cell is selected as a eukaryotic cell. Examples of commonly used eukaryotic cells include fungi (such as Saccharomyces cerevisiae, Saccharomyces cerevisiae, filamentous fungi), insect cells (such as Drosophila S2 or Sf9 cells) and mammalian cells (such as CHO, CHO DG44, CHO-S, COS-7, 293 series cells, HepG2, Huh7, 3T3, RIN, MDCK and HEK293F cell lines).

[0062] Typical vectors include plasmids (e.g., pUC series, pET series, pWB series, pGEX series, pDXW series, pcDNA series, pBR322, pEZ15a, pTZ28a, pMA5, pPICZα, PIC9K, pSET152), viral vectors, bacteriophages (e.g., λgt4λB, λ-Charon, λΔz1, and M13), cosmids, and minichromosomes. Plasmids are the most commonly used vectors, and therefore, in the context of the present invention, unless otherwise specified, plasmids and vectors can be used interchangeably.

[0063] Yet another embodiment of the present invention provides a recombinant cell, wherein the recombinant cell contains the gene sequence encoding the biotin ligase mutant or the biotin ligase expression vector as described above.

[0064] The gene sequence or expression vector can be transformed or transfected into the host cell by various methods known in the art, including: CaCl2 transformation method, calcium phosphate-DNA coprecipitation, electroporation, gene gun bombardment, microinjection, conjugation transfer, liposome-mediated transfection, liposome fusion, lipofection and protoplast fusion, etc.

[0065] In a typical embodiment of the present invention, a mammalian expression system is employed, the expression vector used is pcDNA3.4, and the host cells used are mammalian 293 cell lines, specifically Expi 293F. The gene encoding the biotin ligase mutant is inserted into the multiple cloning site of pcDNA3.4 to construct a Bir A expression vector. Recombinant 293F cells transformed with the expression vector are then obtained. Under the action of the vector's inherent promoter and enhancer, efficient and highly active expression of Bir A is achieved. Furthermore, the vector's inherent selection tags, such as the puromycin resistance gene and the red fluorescent protein gene, facilitate efficient screening of recombinant cells stably expressing Bir A by monitoring their antibiotic resistance and fluorescent protein expression.

[0066] The embodiments of the present invention also provide the use of the biotin ligase mutant, the gene encoding the biotin ligase, the biotin ligase expression vector, or the recombinant cell in preparing biotinylated proteins.

[0067] Biotinylation can be performed in vivo or in vitro. In vitro biotinylation involves co-incubating the target protein with a pure enzyme and biotin under appropriate conditions to achieve biotinylation of the target protein. In vivo biotinylation involves co-transforming host cells with a Bir A expression vector and an expression vector containing a biotin receptor sequence and the target protein. This allows for specific biotinylation of the target protein in vivo while the target protein is expressed.

[0068] Specifically, the in vivo biotinylation comprises the following steps:

[0069] S1. Connect the Avi Tag gene to the upstream or downstream of the target protein gene, insert the fusion gene into the expression vector, and construct the target protein expression vector;

[0070] S2. Transfecting the target protein expression vector and the biotin ligase expression vector into 293F cells to obtain recombinant cells that co-express the target protein and biotin ligase;

[0071] S3. Cultivate the recombinant cells and obtain the biotin-labeled target protein through purification.

[0072] Optionally, the original vectors of the target protein expression vector and the biotin ligase expression vector may be the same or different. Preferably, the same expression vector is used, that is, the target protein expression vector is also pcDNA3.4, so that the two have the same or similar expression efficiency.

[0073] It should be noted that the co-expression in step S2 is only used to indicate that BirA and Avi-target protein fusion proteins coexist in a certain spatiotemporal environment within the recombinant cells, and does not necessarily mean that the expression of the two proteins occurs simultaneously. For example, the Bir A expression vector can be first transformed into the host cells, and then the target protein expression vector can be transformed into the host cells, or vice versa, or both expression vectors can be transfected simultaneously.

[0074] In a preferred embodiment, step S2 comprises: transfecting a biotin ligase expression vector into 293F cells, obtaining a 293F cell line stably expressing biotin ligase through screening, and transfecting a target protein expression vector into a 293F cell line stably expressing biotin ligase to obtain recombinant cells co-expressing the target protein and biotin ligase.

[0075] In step S3, the culture medium used for cell culture is usually supplemented with complex vitamins, including biotin (also known as vitamin H, VH, coenzyme R or vitamin B7, VB7). Therefore, during the recombinant cell culture process, no biotin needs to be added. For example, the serum-free culture medium OPM-293CD05 Medium (purchased from Shanghai Aopumai Biotechnology, product number 81075-001) used in the present invention is used. When there are no vitamins in the culture medium, additional biotin needs to be supplemented as a biotin source for biotinylation of the target protein.

[0076] Biotin easily covalently binds to proteins such as antibodies and is widely used as a common label in various detection systems. In a typical embodiment of the present invention, the biotin-labeled target protein is an antibody for immunoassays. One end of either the light or heavy chain of the antibody is linked to an Avi Tag. The light and heavy chain expression vectors are then transfected into a 293F cell line stably expressing a biotin ligase. Co-expression with the biotin ligase allows the antibody to be expressed in living cells while biotinylation is directly catalyzed by the biotin ligase in vivo. This allows for simultaneous secretory expression and biotin labeling of the antibody, which improves the production efficiency of the biotinylated antibody.

[0077] The present invention will be further described below with reference to specific examples. Experimental methods in the following examples where specific conditions are not specified were generally performed under conventional conditions, such as those described in the Molecular Cloning Laboratory Manual (4th Edition) published by Cold Spring Harbor Laboratory, or under conditions recommended by the manufacturer.

[0078] 1. Screening of biotin ligase mutants and construction of their eukaryotic expression system

[0079] The present invention is based on the wild-type Escherichia coli biotin ligase Bir A (Bifunctional ligase / repressor Bir A, UniProt ID: P06709). By designing different mutation sites at its C-terminus, different forms of Bir A mutants were expressed in eukaryotic cells. By comparing the intracellular Bir A expression level and the enzyme-catalyzed biochemical efficiency of the target protein, biotin ligase mutants with efficient expression in eukaryotic cells and improved enzyme activity were screened. Compared with the wild-type Bir A, the biotin ligase mutants contained the following amino acid substitutions: F271S, Q295L, and A319V.

[0080] The protein sequence of wild-type biotin ligase Bir A is shown below:

[0081] MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRDWGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSGDACIAEYQQQAGRGRRGRKWFSPFGANLYLSM FWRLEQGPAAAIGLSLVIGIVMAEVLRKLGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGIMAMRRVEESVVNQGWITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDN F INRPVKLIIGDKEIFGISRGIDK Q GALLLEQDGIIKPWMGGEISLRS A EK (see SEQ ID NO. 1), the shaded and underlined sites are the sites to be mutated.

[0082] The protein sequence of the biotin ligase Bir A mutant is shown below:

[0083] MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRDWGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSGDACIAEYQQAGRGRRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRK LGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGIMAMRRVEESVVNQGWITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNSINRPVKLIIGDKEIFGISRGIDKLGALLLEQDGIIKPWMGGEISLRSVEK (see SEQ ID NO.2), the mutation site is shown in bold shade.

[0084] 1.1. Construction of biotin ligase expression vector

[0085] The pcDNA3.4 expression vector and mammalian cells Expi 293F (human embryonic kidney cells) were selected as host cells to construct a eukaryotic expression system. The wild-type and mutant Bir A gene sequences fused with a His tag were constructed into the pcDNA3.4 expression vector by total gene synthesis. The fusion gene was inserted into the NdeI site to obtain a biotin ligase expression vector. The vector map is shown below. Figure 1 As shown, the vector was successfully constructed by sequencing.

[0086] The gene sequence of the Bir A mutant is shown below:

[0087] atgaaggataacaccgtgccactgaaattgattgccctgttagcgaacggtgaatttcactctggcgagcagttgggtgaaacgctgggaatgagccgggcggctattaataaacacattcagacactgcgtgactggggcgttgatgtctttaccgttccgggtaaaggatacagcctgcctgagcctatccagttacttaatgctaaacagatattgggtcagctggatggcggtagtgtagccgtgctgcctgtgattgactccacgaatcagtaccttcttgatcgtatcggagagcttaaatcgggcgatgcttgcattgcagaataccagcaggctggccgtggtcgccggggtcggaaatggttttcgccttttggcgcaaacttatatttgtcgatgttctggcgtctggaacaaggcccggcggcggcgattggtttaagtctggttatcggtatcgtgatggcggaagtattacgcaagctgggtgcagataaagttcgtgttaaatggcctaatgacctctatctgcaggatcgcaagctggcaggcattctggtggagctgactggcaaaactggcgatgcggcgcaaatagtcattggagccgggatcaacatggcaatgcgccgtgttgaagagagtgtcgttaatcaggggtggatcacgctgcaggaagcggggatcaatctcgatcgtaatacgttggcggccatgctaatacgtgaattacgtgctgcgttggaactcttcgaacaagaaggattggcaccttatctgtcgcgctgggaaaagctggataacagtattaatcgcccagtgaaacttatcattggtgataaagaaatatttggcatttcacgcggaatagacaaactgggggctttattacttgagcaggatggaataataaaaccctggatgggcggtgaaatatccctgcgtagtgtagaaaaa (see SEQ ID NO.3).

[0088] 1.2 Transformation of Expi 293F cells with biotin ligase expression vector

[0089] The recombinant biotin ligase expression vector was digested with restriction endonuclease SalI (purchased from ABclonal, product number RK21123) to linearize it, thereby obtaining a linearized expression vector.

[0090] The linearized expression vector was transfected into Expi 293F cells using transient transfection technology, including the following steps: diluting the vector DNA with OptiMEM (purchased from Thermo Fisher, cat. no. 31985070) in a sterile test tube so that 1 μg DNA was used per 1 mL of cells to be transfected; in addition, 1 mg / mL of PEI transfection reagent (PEI MAX TM -Transfection Grade Linear Polyethylenimine Hydrochloride (MW 40000) purchased from Polysciences, Catalog No. 24765-1) was added to OptiMEM. The amount of OptiMEM used to dilute the vector DNA and dilute the PEI transfection reagent was 1 / 20 of the total cell volume. The PEI solution was then added to the vector solution at a ratio of PEI (μg): vector DNA (μg) = 3:1. The mixture was immediately mixed and the transfection mixture was added to the Expi 293F cell culture medium. The transfected cells were cultured in OPM-293CD05 Medium (purchased from OPM, Catalog No. 81075-001). After 48 hours of culture, the cells were harvested and replaced with fresh culture medium.

[0091] 1.3. Puromycin resistance screening to obtain stable cell lines expressing Bir A

[0092] The pcDNA3.4 vector carries a red fluorescent protein (mCherry) reporter gene and a puromycin resistance marker gene (PuroR), linked by a self-cleaving peptide 2A. Detecting the mCherry fluorescence signal provides a visual indicator of transfection efficiency and Bir A protein expression. Cells are then cultured in a medium containing puromycin (purchased from beyotime, Catalog No. ST551) to kill negative cells and ultimately enrich for positive cells, establishing a pool of stably transfected cells expressing the Bir A mutant.

[0093] Figure 2The image shows, from left to right, mCherry fluorescence microscopy of Expi293F cells untransfected with the vector, three days after transfection with the Bir A mutant expression vector, and a pool of stably transfected Expi293F cells transfected with the Bir A mutant expression vector and selected for puromycin resistance. As can be seen, the fluorescence signal and resistance selection clearly enriched positive cells transfected with the Bir A mutant, forming a pool of stably transfected cells that efficiently express the Bir A mutant.

[0094] 1.4. Verification of Bir A expression levels in stably transfected cell pools by Western blotting

[0095] The same total number of cells of Expi 293F stably transfected with Bir A wild type and mutant were taken, the cells were lysed, and the electrophoresis samples were prepared and electrophoresed using 4-20% Bis-Tris precast gel (purchased from GenScript, trade name YoungPAGE TM , Catalog No. M00928) was used for electrophoresis, and the expression level of Bir A was detected using rabbit anti-histidine (His) tag antibody conjugated with HRP (Rabbit anti-His-Tag-HRP antibody, purchased from Abclonal, Catalog No. AE104, dilution ratio 1:5000).

[0096] Figure 3 The figure shows immunoblotting results of Expi 293F cell lysates before and after transfection with the mutant Bir A expression vector. Lanes 1-4 show 0.4 mg / mL bovine serum albumin (BSA), a molecular marker, and Expi 293F stably transfected cells with the mutant (Mutant) and unmutated (WT) Bir A expression vectors, respectively. As can be seen from the figure, the optimized Bir A mutant has improved expression efficiency in mammalian cells compared to the wild-type.

[0097] 2. Intracellular biotinylation of recombinant antibodies

[0098] 2.1. Expression and purification of recombinant antibodies containing Avi Tag

[0099] In this example, an antibody was used as the protein to be biotinylated. An antibody heavy chain expression vector with the Avi Tag sequence (GLNDIFEAQKIEWHE) was designed and co-transfected with the light chain expression vector into the aforementioned Bir A stably transfected cells. During recombinant cell culture, Bir A catalyzed the covalent binding of the Avi Tag to biotin, achieving site-specific biotinylation at the lysine residue (K) of the antibody heavy chain Avi Tag to verify the in vivo enzymatic catalytic efficiency of the Bir A mutant. The antibody used was anti-CD52 alemtuzumab, a humanized IgG1 monoclonal antibody that targets and depletes B and T cells by binding to CD52 and has the potential to be used in the study of multiple sclerosis and chronic lymphocytic leukemia.

[0100] The amino acid sequence of the alemtuzumab heavy chain is shown below:

[0101] QVQLQESGPGLVRPSQTLSLTCTVSGFTFTDFYMNWVRQPPGRGLEWIGFIRDKAKGYTTEYNPSVKGRVTMLVDTSKNQFSLRLSSVTAADTAVYYCAREGHTAAPFDY WGQGSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEA PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (see SEQ IDNO.4);

[0102] The amino acid sequence of the light chain of alemtuzumab is shown below:

[0103] DIQMTQSPSSSLSASVGDRVTITCKASQNIDKYLNWYQQKPGKAPKLLIYNTNNLQTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCLQHISRPRTFGQGTKVEIKRTV AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (see SEQID NO.5).

[0104] First, the light and heavy chain genes of the Alemtuzumab antibody were constructed into pCDNA3.4 by whole gene synthesis. The Avi Tag gene sequence was fused to the C-terminus of the heavy chain gene to obtain the expression vectors of the light and heavy chains. The vector map is shown in the figure. Figure 4 As shown, the heavy chain is on the left and the light chain is on the right. The light and heavy chain gene expression vectors were then mixed at a mass ratio of 1:2 and transfected into Expi-293F cells stably expressing the Bir A mutant using the same method as described in "1.2. Transformation of Expi-293F Cells with Biotin Ligase Expression Vectors." A recombinant antibody without the Avi Tag expressed in Expi-293F cells transformed with the Bir A mutant and a recombinant antibody with the Avi Tag expressed in Expi-293F cells transformed with the wild-type Bir A served as controls. Secretory expression of the antibody was driven by the signal peptide provided by the vector.

[0105] Transfected cells were cultured at 37°C, 135 rpm, and 5% CO2. Harvest the culture medium of Expi293F cells 96 hours after transfection, transfer it to a 50 mL centrifuge tube, and centrifuge at 400 g for 10 minutes at 4°C (or room temperature). The pellet was discarded and the supernatant was collected.

[0106] Biotinylated recombinant antibodies were purified using Protein A affinity chromatography. The harvested cell supernatant was incubated with Protein A (purchased from smart-lifesciences, catalog number SA012100) affinity chromatography medium as follows: (1) 0.5 mL of Protein A affinity chromatography medium was taken and centrifuged at 400 g for 2 min at room temperature, and the supernatant was discarded; 3 mL of equilibration buffer was added to resuspend the medium, inverted 4-5 times, and centrifuged at 400 g for 2 min at room temperature, and repeated once; (2) The washed medium was added to the culture supernatant, incubated at 4°C with shaking for 3-4 h, and then centrifuged at 1000 g for 10 min, and the culture medium was carefully aspirated; (3) Ten times the volume of the medium was added to resuspend the medium with wash buffer, centrifuged at 1000 g for 10 min, and the wash buffer was carefully aspirated, and repeated twice; (4) Six times the volume of the medium was added to the elution buffer and transferred to an empty spin column, centrifuged at 1000 g for 2 min to collect the purified CD52 antibody, and the pH was adjusted to neutral using neutralization buffer.

[0107] The buffer formulations mentioned above are as follows: (1) equilibration buffer: 0.02 M phosphate buffer, pH 7.0; (2) elution buffer: 0.2 M citric acid, pH 3.0; (3) neutralization buffer: 0.1 M Tris, pH 9.0.

[0108] The results of recombinant antibody purification are shown in Figure 5 Lanes 1-4 show the reducing (lanes 1-2) and non-reducing (lanes 3-4) electrophoresis images of recombinant antibodies without and with the Avi tag expressed in Expi 293F cells transformed with the Bir A mutant. The results show that the addition of the Avi tag does not affect the normal expression of the antibody in the cells, and its expression efficiency is not affected.

[0109] 2.2. Determination of biotinylation of recombinant antibodies catalyzed by Bir A in cells

[0110] The biotinylation of the purified recombinant antibody was verified by enzyme-linked immunosorbent assay (ELISA) as follows: (1) Coating: 1 μg / mL goat anti-human IgG Fc (purchased from Jackson ImmunoResearch, catalog number 109-005-008) was coated into a 384-well microplate at 25 μL / well and coated overnight at 4°C; (2) Blocking: The coated microplate was removed, the liquid in the wells was removed, and blocking solution (3% skim milk) was added at 50 μL / well and incubated at room temperature for 1 hour; (3) Primary antibody incubation: The purified biotinylated CD52 antibody was used as the primary antibody and diluted 3-fold starting at a starting concentration of 1 μg / mL for a total of 8 dilutions. The antibody dilution solution was diluted at 25 μL / well. The amount of the wells was added to the microplate and incubated at room temperature for 1 hour; NC control was added with 5% PBST diluent; (4) Secondary antibody incubation: remove the liquid in the wells, add HRP-coupled biotin binding protein Neutravidin-HRP at 25 μL / well, and incubate at room temperature in the dark for 1 hour; (5) Color development: add TMB color development solution (purchased from ThermoFisher, product number 34029) at 25 μL / well, and let it stand at room temperature in the dark for 3 minutes. After color development, add stop solution (700 mM oxalic acid solution) to each well to stop the reaction. After the reaction is terminated, place the ELISA plate in a microplate reader and measure the OD value at 450 nm and 630 nm wavelengths. The OD value is expressed as OD. 450nm -OD 630nm The OD correction value is the vertical axis, and the CD52 antibody concentration is the horizontal axis. Draw a binding curve graph, as shown in Figure 6 As shown, the horizontal axis is the CD52 antibody concentration, and the vertical axis is OD 450 and OD 630 The difference in absorbance (OD 450 -OD 630 ).

[0111] ELISA detection found that the Avi Tag-containing antibody expressed in cells stably expressing the Bir A mutant was biotinylated, and the efficiency of the mutated Bir A enzyme in catalyzing the binding of the target protein to biotin was significantly higher than that of the unmutated Bir A, which was reflected in the significantly larger detection high value of the curve.

[0112] In addition, a biotinylated antibody chemically labeled in vitro was used as a control. A 10 mM biotin stock solution was prepared by dissolving 1 mg of activated Sulfo-NHS-LC-biotin (purchased from Thermofisher, Cat. No. 21335) in 180 μL of deionized water on ice. The amount of 10 mM biotin used to label the target protein (CD52 antibody with an Avi-tag) was determined using the following formula: Volume of biotin = 1.34 × (150 / protein MW, calculated as KD) × (protein amount in μg) / 100; the target protein and biotin stock solution were mixed and reacted at 4°C for 2 h.

[0113] ELISA was used to verify the difference in antibody biotinylation efficiency between in vivo enzyme-catalyzed labeling and in vitro chemical labeling. The operation was as follows: (1) Coating: 2 μg / mL Neutravidin protein (purchased from ThermoFisher, product number 31000) was coated into the microplate at 25 μL / well and coated overnight at 4°C. (2) Blocking: The coated microplate was removed, the liquid in the wells was removed, and blocking solution (3% skim milk) was added at 50 μL / well and incubated at room temperature for 1 hour. (3) Primary antibody incubation: Purified biotinylated CD52 antibody was used as the primary antibody and diluted 3-fold starting at a starting concentration of 1 μg / mL for a total of 8 dilutions. The antibody dilution solution was added to the microplate at 25 μL / well and incubated at room temperature for 1 hour. 5% PBST dilution solution was added to the NC control. (4) Secondary antibody incubation: The liquid in the wells was removed and HRP-conjugated monkey anti-human IgG antibody Donkey was added at 25 μL / well. Anti-Human-HRP (purchased from Jackson ImmunoResearch, catalog number 709-035-149), incubate at room temperature in the dark for 1 hour; (5) Color development: TMB color development solution (purchased from Thermo Fisher, catalog number 31000) was added at 25 μL / well, and the reaction was allowed to stand at room temperature in the dark for 5 minutes. After color development, stop solution (700 mM oxalic acid solution) was added to each well to terminate the reaction. After the reaction was terminated, the ELISA plate was placed in a microplate reader and the OD values ​​at 450 nm and 630 nm were measured. 450nm -OD 630nm The OD correction value is the vertical axis, and the CD52 antibody concentration is the horizontal axis. Draw a binding curve graph, as shown in Figure 7 As shown, the horizontal axis is the CD52 antibody concentration, and the vertical axis is OD 450 and OD 630 The difference in absorbance (OD 450 -OD 630 ).

[0114] from Figure 7As can be seen from the results, the Bir A mutants provided by the present invention have high catalytic activity. The efficiency of in vivo biotinylation of target proteins catalyzed by stably transfected cell lines with Bir A mutants is essentially comparable to that of currently commonly used in vitro chemical labeling methods. This facilitates the simplified preparation of biotinylated proteins, enabling simultaneous biotinylation during protein expression, significantly saving time and costs. Furthermore, the need for additional in vitro biotin labeling can also be eliminated, saving material costs.

[0115] 3. Detection of biotin labeling efficiency of recombinant antibodies by biotin ligase in vitro and in vivo

[0116] Bir A and CD52 antibody (with an Avi tag on the H chain) were expressed in Expi 293F cells, respectively. The basic procedures were the same as above and are not repeated here. Bir A enzyme was purified using nickel affinity chromatography, and the biotinylated recombinant antibody was purified using Protein A affinity chromatography. For the procedures, refer to "2.1. Expression and Purification of Avi-Tagged Recombinant Antibodies."

[0117] In vitro enzymatic biotinylation of recombinant antibodies with Bir A mutants: Purified Bir A mutants were incubated with purified CD52 antibody and biotin at 30°C for 1 h. The reaction solution (pH 8.0) contained the antibody, Bir A enzyme, 50 mM N,N-bicine, 10 mM ATP, 10 mM magnesium acetate (MgOAc), and 50 μM D-Biotin. The molar ratio of antibody:enzyme:biotin was 18:1:167. After the reaction, the reaction solution was collected. Wild-type Bir A (WT) was used as a control, and a negative control without Bir A enzyme was also prepared.

[0118] In vivo enzymatic biotinylation of recombinant antibodies catalyzed by Bir A mutants: A target protein expression vector containing an Avi Tag tag was transformed into Bir A stably transfected cells. Transfected cells were cultured in serum-free OPM-293CD05 Medium 81075-001 (Shanghai Aopumai Biotechnology) at 37°C, 135 rpm, and 5% CO2 to achieve site-specific biotinylation in the cells while simultaneously producing the target protein. For related procedures, see "2.1. Expression and Purification of Avi Tag-Tagged Recombinant Antibodies." Wild-type Bir A (WT) was used as a control.

[0119] The HABA Biotin Labeling Efficiency Assay Kit (purchased from beyotime, Cat. No. P0371) was used to assess the biotin labeling efficiency of recombinant proteins. The assay principle is as follows: HABA (4'-hydroxyazobenzene-2-carboxylic acid) is a dye that weakly interacts with streptavidin. Free HABA has a maximum absorbance peak at 356 nm. When HABA binds to streptavidin or avidin to form a complex, a red-shift occurs, with the maximum absorbance peak shifting to 500 nm. Compared to the high affinity of biotin and streptavidin, the affinity of HABA for streptavidin is weak. Therefore, when biotin is present in the solution, it strongly competes with streptavidin for dissociation, resulting in a decrease in absorbance at 500 nm when HABA binds to streptavidin. Therefore, HABA is often used to determine the degree of biotinylation of antibodies or proteins. The absorbance is inversely correlated with the molar amount of biotin in the sample; that is, the lower the absorbance, the more biotinylated the antibody or protein. Therefore, by measuring the absorbance of the HABA-avidin solution before and after adding the biotin-containing sample, the unknown amount of biotin in the solution can be quantified using a standard curve. The detection process is as follows: (1) 160 μL Reaction Buffer and 20 μL HABA / Streptavidin premix (Premix) are added to a 96-well flat-bottom plate in sequence, and gently mixed to avoid foaming; (2) the absorbance value at 500 nm is detected, and this value is recorded as A500 HABA / Streptavidin (A500 H / S); (3) 20 μL of Biotin-BSA (positive control, used to establish a standard curve), Reaction Buffer (negative control) or the sample to be tested (biotinylated protein or biotin-containing sample) are added to different wells of the 96-well flat-bottom plate, and gently mixed to avoid foaming; (4) the absorbance value at 500 nm is detected, and this value is recorded as A500 HABA / Streptavidin / Biotin (A500 H / S / B).

[0120] Calculation of biotin labeling efficiency: (1) Calculate the difference in absorbance at 500 nm of the HABA / Streptavidin Premix before and after adding the sample to be tested: ΔA500 = A500 H / S - A500 H / S / B; (2) According to the Lambert-Beer law (ΔA = ε × c × l, A: absorbance, ε: extinction coefficient, unit is M -1 cm -1, c: concentration of the substance, unit is mol / L, l: optical path length, unit is cm) Calculate the molar concentration of Biotin in the reaction system (mM): Biotin (mM) = Biotin (nm) / Reaction medium (mL) = ΔA500 / 34500M -1 cm -1 × optical path length (cm) (Note 1: The extinction coefficient (ε) of the HABA / Streptavidin complex at 500 nm is 34500 M -1 cm -1 ); (3) Based on the molar ratio of protein concentration (mM) to biotin concentration (mM) in the sample to be tested, calculate the average number of biotin-labeled antibodies or proteins using the formula: biotin (mM) / antibody or protein (mM). The relevant results are shown in Table 1. The concentration of labeled proteins in vivo was calculated using the A280 extinction coefficient method.

[0121] Table 1 Biotinylation efficiency test of wild type and Bir A mutant in vitro and in vivo

[0122]

[0123]

[0124] As can be seen from Table 1, the Bir A mutant can catalyze the labeling of an average of 1.83 biotin molecules per antibody in vivo, which is similar to the catalytic efficiency of the enzyme in vitro, indicating that the Bir A mutant has high expression and catalytic activity in the eukaryotic expression system. Compared with the wild-type Bir A, its expression efficiency and biotinylation catalytic activity for the target protein are significantly improved, and the labeling efficiency is significantly better than that of the non-mutated Bir A.

[0125] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A biotin ligase mutant, characterized in that The amino acid sequence is shown in SEQ ID NO.

2.

2. A gene sequence encoding the biotin ligase mutant according to claim 1.

3. The gene sequence encoding the biotin ligase mutant according to claim 2, characterized in that: The gene sequence is shown in SEQ ID NO.

3.

4. A biotin ligase expression vector, characterized in that: The expression vector comprises the gene sequence encoding the biotin ligase mutant according to any one of claims 2 to 3.

5. The biotin ligase expression vector according to claim 4, characterized in that The expression vector is pcDNA3.

4.

6. A recombinant cell, characterized in that The recombinant cell comprises the gene sequence encoding the biotin ligase mutant according to any one of claims 2 to 3 or the biotin ligase expression vector according to any one of claims 4 to 5.

7. The recombinant cell according to claim 6, characterized in that The recombinant cells are 293F cells.

8. Use of the biotin ligase mutant according to claim 1, the gene sequence encoding the biotin ligase mutant according to any one of claims 2-3, the biotin ligase expression vector according to any one of claims 4-5, or the recombinant cell according to any one of claims 6-7 in preparing a biotinylated protein.

9. Use of the biotin ligase mutant, the gene sequence encoding the biotin ligase mutant, the biotin ligase expression vector or the recombinant cell according to claim 8 in preparing a biotinylated protein, characterized in that: The biotinylated protein can be prepared in vivo or in vitro.

10. Use of the biotin ligase mutant, the gene sequence encoding the biotin ligase mutant, the biotin ligase expression vector or the recombinant cell according to claim 9 in preparing biotinylated protein, characterized in that: The in vivo preparation of the biotinylated protein comprises the following steps: S1. Connect the Avi Tag gene to the upstream or downstream of the target protein gene, insert the fusion gene into the expression vector, and construct the target protein expression vector; S2. Transfecting the target protein expression vector and the biotin ligase expression vector into 293F cells to obtain recombinant cells that co-express the target protein and biotin ligase; S3. Cultivate the recombinant cells and obtain the biotin-labeled target protein through purification.

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