Method for efficiently preparing fixed-point biotinylation recombinant protein in escherichia coli body

Abstract

The invention discloses a method for efficiently preparing fixed-point biotinylation recombinant protein in escherichia coli, and belongs to the technical field of biology. According to the invention, a weakened expression regulatory sequence regulates and encodes a biotin ligase (BirA) gene expression cassette and a target protein gene expression cassette encoding a sequence carrying a biotin receptor peptide tag (AVI-Tag) to be jointly constructed on an escherichia coli expression vector, so that efficient fixed-point biotinylation of a target protein in an escherichia coli body is realized. The method has the advantages of high biotinylation efficiency, high specificity, simplicity and convenience in operation and the like, can be widely applied to the fields of separation and purification of proteins, protein interaction research and the like, and has important practical application value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a method for efficiently preparing site-specific biotinylated recombinant proteins in vivo in Escherichia coli. BACKGROUND

[0002] Biotinylation technology is widely used in the field of molecular biology, and the core reason lies in the fact that there is a strong and specific interaction between biotin and streptavidin. The binding strength of the two is much higher than that of antigen-antibody binding reaction, and they almost do not non-specifically bind to other biological molecules, which can accurately target target molecules in complex biological samples and reduce background interference. At the same time, the stability of biotin and streptavidin after binding is very strong and is not easily affected by extreme conditions.

[0003] Biotin ligase is a bifunctional protein encoded by the BirA gene of Escherichia coli, which has unique molecular recognition characteristics. The enzyme not only can specifically recognize a specific amino acid sequence in a protein molecule and catalyze the covalent binding of biotin to the target protein, but also can act as a transcriptional repressor protein to regulate the expression of biotin synthesis-related operons. AVI-Tag is a short peptide composed of 15 amino acids, and its sequence is GLNDIFEAQKIEWHE. It can be specifically recognized and catalyzed by biotin ligase to covalently bind biotin molecules to specific lysine residues on AVI-Tag, so that the protein with AVI-Tag is labeled with biotin. This site-specific labeling method not only realizes the specific biotinylation of proteins, but also significantly improves the binding specificity and affinity of the avidin-biotin system. At present, this technology has shown important application value in the research fields of protein purification, protein interaction screening, and fluorescent labeling.

[0004] Escherichia coli is a commonly used genetic engineering host bacterium, which has the advantages of fast reproduction speed, low culture cost, clear genetic background, and perfect gene editing tools. It is the main host for recombinant protein expression, and the endogenous metabolic network can also provide the necessary substrate and enzyme environment for biotin synthesis and transfer. However, the biotinylation achieved by the existing technology in Escherichia coli mainly depends on in vitro labeling or non-site-specific intracellular modification. In vitro labeling requires the purification of target proteins before chemical or enzymatic reaction, which is not only complicated and costly, but also may cause protein denaturation during the purification process. Traditional non-site-specific intracellular modification often modifies non-expected sites of host cell proteins or target proteins, resulting in non-uniform products and affecting the efficiency and specificity of subsequent purification, which limits its application in actual research and production.

[0005] Therefore, it is an important topic to develop an efficient site-specific biotinylation method for recombinant proteins in vivo in Escherichia coli. SUMMARY

[0006] To overcome the problems of low specificity and low efficiency of the in vivo biotinylation system of E. coli in the prior art, the present application aims to provide a method for efficiently preparing site-specific biotinylated recombinant proteins in E. coli. In the present application, a weakly expressed biotin ligase gene expression frame and a gene expression frame encoding a target protein carrying an AVI-Tag sequence are co-expressed on an E. coli expression vector to realize the simultaneous site-specific modification of the target protein during its synthesis in E. coli. This "in situ precise labeling" mode has multiple advantages: on the one hand, the modification process is synchronized with protein synthesis, eliminating the need for in vitro operation, which simplifies the process and avoids the loss of activity caused by post-purification modification; on the other hand, the high specificity of AVI-Tag and biotin ligase ensures that the modification only occurs at the target site, without affecting the structure and function of the protein, and ensures that each target protein carries an equal amount of biotin, and the product is uniform.

[0007] To achieve the above-mentioned object, the present application provides the following technical solutions: In a first aspect, the present application provides a method for efficiently preparing site-specific biotinylated recombinant proteins in E. coli, comprising the following steps: (1) obtaining a fusion gene fragment encoding AVI-Tag-target protein and a gene fragment encoding biotin ligase; (2) inserting the gene fragment encoding AVI-Tag-target protein and the gene fragment encoding biotin ligase in step (1) into different multiple cloning sites of the same expression vector to construct a recombinant expression vector; (3) transforming the recombinant expression vector obtained in step (2) into E. coli BL21(DE3); (4) expanding the culture of the recombinant strain obtained in step (3) and inducing the expression of the recombinant protein, breaking the bacterial cells, and obtaining the biotinylated recombinant protein by nickel column purification of the protein supernatant.

[0008] Further, the AVI-Tag in step (1) is a short peptide composed of 15 amino acids, and its amino acid sequence is shown in SEQ ID NO. 7.

[0009] Further, the biotin ligase in step (1) is BirA derived from Bacillus subtilis, and its amino acid sequence is shown in SEQ ID NO. 6, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO. 5.

[0010] Further, the amino acid sequence of the AVI-Tag-target protein in step (1) is shown as SEQ ID NO. 4, and the nucleotide sequence of the fusion gene fragment encoding the AVI-Tag-target protein is shown as SEQ ID NO. 3.

[0011] Further, the fusion gene fragment encoding the AVI-Tag-target protein in step (2) is regulated by a T7 promoter, and the nucleotide sequence of the T7 promoter is shown as SEQ ID NO. 2.

[0012] Further, the gene fragment encoding the biotin ligase in step (2) is regulated by a weakened expression regulatory sequence, and the nucleotide sequence of the weakened expression regulatory sequence is shown as SEQ ID NO. 1.

[0013] Further, the expression induction condition in step (4) is 25-38℃, 100-300 rpm, and the expression is induced for 12-24 hours in an auto-induction medium.

[0014] Further, the purification process in step (4) is as follows: the protein supernatant is passed through a column, and PBS buffer containing 19-21 mM imidazole is used to pass through the column to remove non-specifically adsorbed impurities, and then PBS buffer containing 40 mM, 60 mM, 80 mM, 120 mM, 240 mM, and 500 mM imidazole is used to elute the target protein.

[0015] In a second aspect, the present application provides a site-specific biotinylated recombinant protein prepared by the method described above.

[0016] Compared with the prior art, the present application has the following beneficial effects: The method has high conversion efficiency: the biotin ligase gene and the target protein gene can be introduced into the host cell only once by using a single plasmid system, the cumbersome operation of twice transformation in a double plasmid system is avoided, and the conversion efficiency is significantly improved. The method has good gene expression coordination: the biotin ligase gene and the target protein gene carrying the AVI-Tag sequence on the same plasmid have the same copy number in the host cell, are affected by the intracellular environment, and have coordinated expression, so that the biotinylation reaction is efficiently performed. The method is simple in operation and cost-saving: the advantages of Escherichia coli, such as short growth cycle, low culture cost and simple genetic operation, are used to efficiently express the recombinant protein in the bacteria and realize in-vivo biotinylation modification, so that the risk of protein loss is reduced, and non-specific site labeling and expensive reagents required by in-vitro chemical modification are avoided. The method has high biotinylation efficiency and strong specificity: the biotin ligase encoded by the BirA gene selected in the application has high catalytic activity, can efficiently transfer biotin to the AVI-Tag sequence of the target protein, greatly improves the biotinylation efficiency, and further optimizes the biotinylation efficiency through the differential design of the promoter; the AVI-Tag sequence has high specificity, avoids non-specific biotinylation, and improves the specificity of biotinylation. The method has wide application: can be used in protein separation and purification, protein interaction research, protein localization and tracking and other fields, and has important practical application value. BRIEF DESCRIPTION OF DRAWINGS In order to more clearly illustrate the embodiments of the application, the drawings involved in the embodiments will be briefly introduced below.

[0017] Figure 1 It is a schematic diagram of the gene structure design of BirA (A) and AVI-mBaoJin (B) recombinant proteins.

[0018] Figure 2 It is a gene map of pAVI-BaoJin-BirA plasmid.

[0019] Figure 3 It is an expression and purification result diagram of biotinylated recombinant protein AVI-mBaoJin, wherein M: pre-stained protein Marker, 1: purified protein; 2: whole protein before induction; 3: whole protein after induction; 4: soluble protein after induction; 5: insoluble protein after induction.

[0020] Figure 4 It is a biotinylation Western Blot verification result diagram of biotinylated recombinant protein AVI-mBaoJin, wherein M: pre-stained protein Marker, 1: purified protein; 2: whole protein after induction; 3: soluble protein after induction; 4: insoluble protein after induction.

[0021] Figure 5 Figure for biotinylated recombinant protein AVI-mBaoJin biotinylation efficiency verification results, wherein M: pre-stained protein marker, 1-2: 2 μg purified protein; 3: 2 μg purified protein supernatant after binding with streptavidin magnetic beads; 4: first wash solution after binding with streptavidin magnetic beads; 5: second wash solution after binding with streptavidin magnetic beads. DETAILED DESCRIPTION

[0022] The application will be described in detail below with reference to the embodiments, but the embodiments of the application are not limited thereto. It is obvious that the embodiments described below are only part of the embodiments of the application, and other similar embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the application.

[0023] Example 1: Design of biotinylated recombinant protein The gene encoding AVI-Tag was fused with the target gene mBaoJin (synthesized by commissioning Jiutian Gene Technology Co., Ltd.), and AVI-Tag can be recognized by biotin ligase (BirA) and covalently combined with endogenous biotin synthesized by E. coli itself, so that the recombinant protein AVI-mBaoJin carries a biotin tag.

[0024] The biotin ligase gene and the AVI-Tag fusion target protein recombinant gene are respectively regulated by independent promoters, wherein the promoter regulating the biotin ligase gene is a weakened expression regulation sequence, and the promoter regulating the target protein gene is a strong T7 promoter; the recombinant expression vector gene structure is as shown in Figure 1 According to the design, the biotinylated protein recombinant plasmid pAVI-BaoJin-BirA was synthesized, and the plasmid gene map is as shown in Figure 2 .

[0025] The weakened expression regulation sequence is as shown below (SEQ ID NO. 1): tttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgaattctagataacgagggcaaaaa The nucleotide sequence of the strong T7 promoter is as shown below (SEQ ID NO. 2): taatacgactcactatagg The nucleotide sequence encoding AVI-mBaoJin is as shown below (SEQ ID NO. 3): atgggactaaatgatatatttgaagctcaaaagatcgaatggcatgaaggtggtggccatcaccaccaccaccactccggcggcggcggccaccaccaccaccaccacagtggcggcggcggtatggtttcaaagggcgaagaggagaacatggcttccaccccgtttaaattccaactgaagggtacgatcaatggtaagagcttcactgtagagggcgagggtgaggggaacagccacgaaggtagccacaagggcaagtacgtttgtaccagcggtaaactgccgatgagctgggcggcactgggtacgacctttggttatggtatgaagtactataccaaatatccgagcggtctgaaaaattggtttcgtgaggttatgccgggtggctttacctatgatcgtcatatccagtacaaaggtgacggcagcattcatgcgaaacaccaacatttcatgaagaacgggacgtaccataacatcgtggagttcacgggccaagatttcaaagagaactccccggtcctgaccggtgacatgaatgtgagcttgccaaatgaagtgccgcagattccgcgtgacgacggtgtggagtgcccggtcaccctgttgtacccactgctgtccgataaaagcaagtacgttgaggctcatcaatataccatttgcaaaccgttacacaaccagccggcccctgatgttccgtatcattggattcgcaaacagtacacccagagcaaagacgacgcggaagagcgcgatcatatctgccagtctgaaaccttggaagcgcatctgaagggcatggatgaactctacaagggtggtggtggttcgagctcgggc The amino acid sequence of AVI-mBaoJin is shown below (SEQ ID NO. 4): MGLNDIFEAQ KIEWHEGGGH HHHHHSGGGG HHHHHSGGGG MVSKGEEENM ASTPFKFQLK GTINGKSFTVE GEGEGNSHEG SHKGKYVCTS GKLPMSWAAL GTTFGYGMKY YTKYPSGLKN WFREVMPGGF TYDRHIQYKGD GS1HAKHQHF MKNGTYHNIV EFTGQDFKEN SPVLTGDMNV SLPNEVPQIP RDDGVECPVT LLYPLLSDKS KYVEAHQYTI CKPLHNQPAP DVPYHWIRKQ YTQSKDDAEER DHICQSETLE AHLKGMDELY KGGGGSSSG The nucleotide sequence encoding the biotin ligase is shown below (SEQ ID NO. 5): atgcccatacaactattaaatgctaaacagatcctgggtcagctggacggcggtagcgtggcagttttgccggttattgatagcaccaaccaatatttacttgatcgtattggtgagttgaaaagcggtgacgcctgcatcgccgagtaccagcaggcaggccgtggtcgccgcggccgcaagtggttcagcccgtttggtgctaatttgtacctgtctatgttctggcgtctggagcaaggtccggcggctgcgattggcctgtccctggtgatcggcatcgtgatggcggaagttctgcgtaagctgggcgctgataaagttcgtgtgaagtggccaaatgatctgtacctgcaagatcgcaagcttgcgggcattctggttgagttgacgggaaaaaccggtgacgcggcgcagattgtaatcggcgcgggcatcaacatggctatgcgtcgtgtcgaggaatccgtggtcaatcagggttggattactctgcaagaggcgggcatcaacctggaccgcaacaccctggcggctatgctgattcgtgaattgcgcgcagcgctggaattgtttgaacaagaaggtctggcaccgtatctctctcgttgggaaaaactcgacaactttatcaaccgtccggtgaaattaatcatcggtgataaagaaattttcggcattagccgtggtatcgacaagcagggtgccctgctgttggagcaggacggcatcattaaaccgtggatgggtggtgagatcagcttgagatcggcggagaag The amino acid sequence of the biotin ligase is shown below (SEQ ID NO. 6): MPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSGDACIAEYQQAGRGRRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRKLGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQGWITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIGDKEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK The amino acid sequence of AVI-Tag is shown as follows (SEQ ID NO. 7): GLNDIFEAQKIEWHE.

[0026] Example 2 Expression, purification and biotinylation identification of biotinylated recombinant protein (1) Preparation of chemical competent cells of E. coli BL21 (DE3) I. The single colony of BL21 (DE3) was streaked and activated in 5 mL of LB liquid medium at 37°C, 220 rpm overnight; II. The next day, the bacterial liquid was inoculated at 1:100, and cultured at 37°C, 220 rpm until the OD 600nm value was 0.4-0.6 in the logarithmic phase, and was placed on ice for 30 min; III. The bacterial liquid in the flask was poured into a bacteria collection bottle in a clean bench, centrifuged at 4°C, 4000 rpm for 8 min, the supernatant was discarded, and 50 mL of 0.1 M CaCl2 liquid was added to resuspend the bacterial body; IV. After being placed on ice for 10 min, the bacterial body was centrifuged at 4°C, 5000 rpm for 8 min, the supernatant was discarded, and 50 mL of 0.1 M CaCl2 liquid was added to resuspend the bacterial body; V. After being placed on ice for 10 min, the bacterial body was centrifuged at 4°C, 5000 rpm for 8 min, the supernatant was discarded, and 600 μL of 0.1 M CaCl2 was added to resuspend the bacterial body, an equal volume of 20% glycerol was added, and was aliquoted into 1.5 mL centrifuge tubes and stored at -80°C.

[0027] (2) Chemical transformation of recombinant plasmid The recombinant plasmid pAVI-BaoJin-BirA was transformed into E. coli BL21 (DE3): 1 μL of plasmid with a concentration of 100 ng / μL was taken, the tip was inserted into the bottom, and 50 μL of E. coliMix gently, and incubate on ice for 20 min. Place the centrifuge tube in a 42°C water bath for 80 s, and then incubate on ice for 5 min. Add 200 μL of preheated SOC medium at 37°C, and incubate at 37°C for 30 min. Transfer to a shaker at 37°C and 220 rpm for 1 h. Take 100 μL of the cultured bacterial solution and evenly spread on an LB-Kan plate. Incubate in a 37°C incubator overnight.

[0028] (3) ZY automatic induction expression Take a single colony from the above culture dish and inoculate into 3 mL of LB liquid medium containing 50 μg / mL Kan, and incubate at 37°C and 220 rpm overnight. The next day, take 500 μL of fresh bacterial solution and inoculate into 50 mL of automatic induction medium (see Table 1 for component formula) containing 50 μg / mL kanamycin, and induce expression at 25°C for 22-24 h. The next day, centrifuge at 4°C and 8000 rpm for 10 min, discard the supernatant, and resuspend the bacterial cells with about 20 mL of 0.02M PBS buffer. Centrifuge at 4°C and 8000 rpm for 10 min, discard the supernatant, and resuspend the bacterial cells with 25 mL of 0.02M PBS buffer containing 0.5M NaCl. Store at -80°C.

[0029] (4) Recombinant protein purification I. Freeze-thaw the bacterial solution stored at -80°C at 37°C and -80°C for 3 times to break most of the bacterial cells. Add lysozyme at a final concentration of 50 μg / mL according to the volume of the bacterial solution, and incubate at 37°C for 30 min to allow the lysozyme to work; II. Incubate on ice for 15 min, and sonicate in an ice bath for 10 min (conditions: sonicate for 2 s, pause for 3 s, power 200 W). After completing the sonication, mix well by turning the tube upside down. Adjust the number of times and time of sonication according to the state of the bacterial solution to ensure that the bacterial cells are fully broken; III. Centrifuge at 4°C and 12000 rpm for 10 min, discard the precipitate, and transfer the supernatant to a new tube. Repeat the centrifugation until there is no obvious precipitate at the bottom of the tube. Add an equal volume of PBS buffer containing 40 mM imidazole, mix well, and use a 0.45 μm filter to filter the supernatant to remove impurities and prevent the chromatography column from being blocked. Then add Tween-20 at a final concentration of 3% and mix gently; IV. Equilibration: After using ddH2O to exhaust the air in the sample tube, connect it to the nickel column. Equilibrate the column with 25 mL of ddH2O at a flow rate of 2 mL / min, and then equilibrate it with 20 mL of equilibration liquid (PBS buffer containing 20 mM imidazole + 3% Tween-20) at a flow rate of 1 mL / min. This allows the filler to be in the same buffer system as the target protein, which protects the protein. V. Loading: After equilibration of the column, the ice-cold protein sample was added to the column to allow the sample to contact the packing material and improve the recovery of the target protein. The flow rate was 0.8 mL / min. The flow-through was collected. VI. Impurity removal: The column was washed with 20 mL of PBS buffer containing 20 mM imidazole at a flow rate of 2 mL / min to remove impurities. VII. Elution: The column was eluted with PBS buffer containing 40 mM, 60 mM, 80 mM, 120 mM, 240 mM, and 500 mM imidazole, respectively, at a flow rate of 1.2 mL / min. Each gradient elution fraction, which contained the target protein, was collected and stored at 4°C.

[0030] VIII. The column was washed with 20 mL of PBS buffer containing 1 M imidazole at a flow rate of 2 mL / min, followed by 20 mL of ddH2O at a flow rate of 2 mL / min. Finally, the column was sealed with 20% ethanol.

[0031] (5) Determination of the purity of the recombinant protein A small amount of 80 μL of the induced expression and purified protein sample was mixed with 20 μL of 5×Loading Buffer. After boiling at 99°C for 10 min, the sample was loaded onto an SDS-PAGE gel for electrophoresis. The molecular weight and purity of the purified protein were determined by the electrophoresis results, as shown in Figure 3 .

[0032] (6) Biotinylation identification of the recombinant protein I. Electrophoresis: The treated sample was loaded onto an SDS-PAGE gel for electrophoresis separation; II. Membrane transfer: The proteins in the gel were transferred to a PVDF membrane by electroblotting; III. Blocking: The PVDF membrane was placed in blocking solution and blocked on a shaker at room temperature for 1-2 hours to block non-specific binding sites on the membrane. Then, the membrane was washed with TBST for 5-6 times, each time for 5 minutes; IV. Incubation: The diluted horseradish peroxidase-labeled streptavidin (SA-HRP) solution was evenly applied to the PVDF membrane at a ratio of 1:2000, and incubated on a shaker at room temperature for 30 minutes to 1 hour. The membrane was washed thoroughly with TBST for 4-5 times, each time for 5 minutes, to completely remove the unbound SA-HRP; V. Color development and imaging: According to the instructions of the Super Sensitive Chemiluminescence Kit (Sparkjade ECL star), the A and B solutions were mixed in proportion and evenly dropped onto the membrane. The reaction was carried out in the dark for 1-5 minutes. The chemiluminescence imaging instrument was used to detect and record the luminescence signal, as shown in Figure 4 . The biotinylation of the recombinant protein was identified by the development results.

[0033] Table 1 Components of 1500 mL auto-induction medium

[0034] *1. 50x5052: ① 2.5g glucose, ② 10g α-lactose, ③ 73ml RO water, ④ 25g glycerol, prepare by adding ①, ②, ③ in sequence, 40℃ water bath, intermittent stirring with a small glass rod until the solution becomes clear and transparent, then add 25g glycerol and mix well, and filter for use.

[0035] *2. 20xNPS: ① prepare 160ml 1M Na2HPO4 solution; ② prepare 50ml 1M KH2PO4 solution; ③ measure the pH of the above Na2HPO4 solution, then adjust the pH of the Na2HPO4 solution to 7.2~7.5 using the KH2PO4 solution, if the pH <7.2, add Na2HPO4, if the pH >7.2, add KH2PO4; ④ add ammonium sulfate, calculate the amount of ammonium sulfate to be added (V is the total volume of the solution after step ③) by V*0.5mmol / L*132.14g / ml; ⑤ measure the pH of the solution again, and prepare for use under high pressure.

[0036] *3. ZY culture solution: 4g tryptone, 2g yeast extract, RO water to 400ml, prepare for use under high pressure.

[0037] Example 3 Verification of biotinylation efficiency of recombinant protein I. Resuspend the streptavidin magnetic beads thoroughly; use a pipette to transfer 20 μL of excess streptavidin magnetic beads to a new centrifuge tube, place the centrifuge tube on a magnetic separator, and stand for 1 min (this operation is hereinafter referred to as magnetic separation); use a pipette to remove the supernatant, and remove the centrifuge tube from the magnetic separator.

[0038] II. Add 1 mL of 0.05% PBST to the centrifuge tube, cover the centrifuge tube cap, resuspend the streptavidin magnetic beads thoroughly, magnetically separate, and remove the supernatant.

[0039] III. Repeat step II twice, for a total of 3 washes.

[0040] IV. Add 2 μg of biotinylated recombinant protein obtained by the above method in Example 2 to the streptavidin magnetic beads for co-incubation, resuspend the streptavidin magnetic beads thoroughly; place the centrifuge tube on a rotary mixer, rotate at 20 rpm at room temperature for 60 min to mix the streptavidin magnetic beads and the biotinylated recombinant protein thoroughly.

[0041] V. Magnetically separate, and transfer the supernatant to a new centrifuge tube.

[0042] VI. The streptavidin magnetic beads are washed according to the method of step II. The washing solution is transferred to a new centrifuge tube.

[0043] VII. The target protein not incubated with streptavidin magnetic beads, the supernatant obtained above and the washing solution are loaded into a SDS-PAGE gel for electrophoresis, and the efficiency of biotinylated protein is identified by the electrophoresis result, as shown in Figure 5 Figure 2, no target protein band appears in the supernatant, indicating that the biotinylated protein is completely combined with the streptavidin magnetic beads, and no target protein band appears in the washing solution, indicating that the biotinylated protein has strong binding force with the streptavidin magnetic beads, confirming that the biotinylation efficiency of the target protein reaches 100%.

[0044] The present application designs a recombinant protein, the promoter of the biotin ligase gene is a weakened expression regulatory sequence, and the promoter of the target protein gene is a strong T7 promoter, effectively balancing the expression amount of the biotin ligase and the target protein, successfully inducing expression and purifying AVI-mBaoJin biotinylated recombinant protein, and confirming that an efficient E. coli in vivo site-directed biotinylated recombinant protein is successfully constructed by verifying the expression amount of the target protein and the biotinylation efficiency.

[0045] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application, and not to limit them; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacement for part or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A method for site-directed biotinylation of recombinant proteins in *Escherichia coli*, characterized in that, Includes the following steps: (1) Obtain the fusion gene fragment encoding the AVI-Tag-target protein and the gene fragment encoding biotin ligase; (2) Insert the gene fragment encoding AVI-Tag-target protein and the gene fragment encoding biotin ligase from step (1) into different multiple cloning sites of the same expression vector to construct a recombinant expression vector; (3) Transform the recombinant expression vector obtained in step (2) into E. coli In BL21(DE3); (4) The recombinant strain obtained in step (3) is expanded and induced to express recombinant protein. The bacterial cells are broken, and the resulting protein supernatant is purified by nickel column to obtain biotinylated recombinant protein.

2. The method according to claim 1, characterized in that, The AVI-Tag in step (1) is a short peptide consisting of 15 amino acids, the amino acid sequence of which is shown in SEQ ID NO.

7.

3. The method according to claim 1, characterized in that, The biotin ligase in step (1) is BirA derived from Bacillus subtilis, whose amino acid sequence is shown in SEQ ID NO.6 and whose nucleotide sequence encoding the gene is shown in SEQ ID NO.

5.

4. The method according to claim 1, characterized in that, The amino acid sequence of the AVI-Tag-target protein in step (1) is shown in SEQ ID NO.4, and the nucleotide sequence of the fusion gene fragment encoding the AVI-Tag-target protein is shown in SEQ ID NO.

3.

5. The method according to claim 1, characterized in that, In step (2), the fusion gene fragment encoding the AVI-Tag-target protein is regulated by the T7 promoter, the nucleotide sequence of which is shown in SEQ ID NO.

2.

6. The method according to claim 1, characterized in that, In step (2), the gene fragment encoding biotin ligase is regulated by a weakened expression regulatory sequence, the nucleotide sequence of which is shown in SEQ ID NO.

1.

7. The method according to claim 1, characterized in that, In step (4), the induction conditions are: induction in an auto-induction medium at 25-38°C and 100-300 rpm for 12-24 hours.

8. The method according to claim 1, characterized in that, The purification process described in step (4) is as follows: pass the protein supernatant through a column, pass it through a PBS buffer containing 19-21 mM imidazole to remove non-specifically adsorbed impurities, and elute the target protein sequentially with PBS buffers containing 40 mM, 60 mM, 80 mM, 120 mM, 240 mM, and 500 mM imidazole.

9. The site-directed biotinylated recombinant protein prepared by the method of any one of claims 1-8.

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