A method for expressing BirA enzyme in prokaryotic escherichia coli and purifying the same
By expressing and purifying BirA enzyme in Escherichia coli, the problems of nonspecificity and high cost of chemical labeling methods have been solved, achieving efficient and economical preparation and purification of BirA enzyme, which is suitable for subsequent protein biotinylation labeling.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- CHONGQING MEDICAL UNIVERSITY
- Filing Date
- 2026-04-16
- Publication Date
- 2026-05-29
AI Technical Summary
In existing technologies, chemical labeling methods result in nonspecific protein biotinylation modifications, which may lead to reduced functional activity. Furthermore, commercially available high-quality BirA enzymes are expensive, making it difficult to achieve efficient and economical in vitro biotinylation labeling.
Using a prokaryotic Escherichia coli expression system, the pET-28A-BirA plasmid was constructed and BirA enzyme was induced to be expressed in BL21(DE3). The BirA enzyme was purified by nickel column affinity chromatography using an 8× histidine tag. The induction conditions were optimized to reduce inclusion body formation and improve soluble expression.
This method enables the preparation of high-purity, high-activity BirA enzymes, reduces costs, and is suitable for large-scale preparation. Furthermore, the one-step purification process ensures the enzyme's high catalytic efficiency and the accuracy of its application.
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Abstract
Description
Technical Field
[0001] This invention relates to the fields of genetic engineering and protein engineering technology, and in particular to a method for expressing BirA enzyme in prokaryotic Escherichia coli and purifying it. Background Technology
[0002] The interaction between biotin (also known as vitamin B7 or vitamin H) and avidin and streptavidin is one of the strongest non-covalent interactions known in nature. This super affinity not only has an extremely fast binding rate but also exhibits remarkable stability—the biotin-streptavidin complex remains stable even under extreme conditions such as high temperature, 6 M guanidine hydrochloride, or 1% SDS.
[0003] The biotin-avidin system has a wide range of applications, primarily serving three functions: First, as a fixation tool, it anchors biotin-labeled proteins or nucleic acids onto streptavidin-coated solid supports (such as magnetic beads, ELIS plates, and chips). Second, as a signal amplification element, one streptavidin molecule can simultaneously bind four biotin molecules, enabling precise detection of even low-abundance antigens via biotin-labeled enzymes or fluorophores. Third, as a bridging element, it is used to assemble biotin-labeled proteins, nucleic acids, or small molecule drugs.
[0004] A key common step in these widespread applications is the biotinylation of the target protein. Traditional labeling methods primarily rely on chemical modification, which utilizes activated biotin derivatives (such as NHS-biotin) to react with the ε-amino group of the N-terminal α-amino or lysine residues of proteins, achieving covalent linkage of biotin. The advantages of chemical labeling are its simplicity, lack of need for genetic modification of the target protein, and applicability to rapid labeling of various proteins. However, this method has inherent limitations: since most proteins have multiple lysine residues distributed on their surface, chemical labeling is often random and nonspecific, potentially leading to over-biotinylation at multiple sites. This heterogeneous and uncertain labeling may result in reduced protein functional activity.
[0005] Compared to chemical labeling methods, enzymatic biotinylation offers a more precise alternative. This method utilizes a biotin-protein ligase (BirA) derived from *E. coli* to achieve site-specific biotin modification. BirA, encoded by the *birA* gene in *E. coli*, is a single-subunit enzyme with a molecular weight of approximately 35-37 kDa. The Avi Tag is a short peptide tag, only 15 amino acids in length. BirA can highly specifically recognize lysine residues in the AviTag sequence and, in the presence of ATP and magnesium ions, catalyze the covalent linking of biotin to the ε-amino group of this lysine residue. This enzymatic reaction produces products with good uniformity and high labeling efficiency, enabling quantitative site-specific biotinylation.
[0006] Obtaining high-purity, high-activity BirA enzyme protein is a necessary prerequisite for achieving in vitro biotinylation labeling of target proteins. Currently, although high-quality recombinant BirA products are commercially available, their price is relatively high. Therefore, establishing a laboratory-owned BirA prokaryotic expression and purification system can not only significantly reduce experimental costs but also flexibly adjust the expression scale and purification strategy according to research needs, providing a stable and economical enzyme source for subsequent continuous in vitro biotinylation applications.
[0007] The *E. coli* expression system has become the preferred host for recombinant BirA enzyme expression due to its advantages such as clear genetic background, simple operation, short culture cycle, and high protein expression levels. This study aims to clone the BirA gene from the *E. coli* genome, construct a prokaryotic expression vector, and induce recombinant BirA protein expression in *E. coli* BL21(DE3). High-purity enzyme protein will be obtained through affinity purification. This will provide a high-quality enzyme tool for subsequent protein site-specific labeling and functional studies based on the Avi Tag / BirA system. Summary of the Invention
[0008] The purpose of this invention is to address the shortcomings of existing technologies by proposing a method for expressing BirA enzyme in Escherichia coli and purifying it.
[0009] To achieve the above objectives, the present invention adopts the following technical solution: A recombinant expression vector for expressing BirA enzyme, wherein the recombinant expression vector is a pET-28A-BirA plasmid constructed by inserting a BirA enzyme encoding gene into the multiple cloning site of the pET-28A vector; the C-terminus of the BirA enzyme encoding gene is fused with a nucleotide sequence encoding an 8×histidine tag.
[0010] Preferably, the nucleotide sequence of the BirA enzyme encoding gene is SEQ ID NO:1.
[0011] A recombinant Escherichia coli engineered bacterium expressing BirA enzyme, said engineered bacterium comprising the above-described recombinant expression vector.
[0012] Preferably, the host bacterium of the engineered bacteria is Escherichia coli BL21(DE3).
[0013] A method for recombinant expression of BirA enzyme includes the steps of culturing the above-mentioned recombinant Escherichia coli engineered bacteria and inducing the expression of the BirA enzyme.
[0014] Preferably, IPTG is used as the inducing agent for the induced expression; the final induction concentration of IPTG is 0.5 mM.
[0015] Preferably, the induction temperature is 18°C and the induction time is 5-6 hours.
[0016] A method for purifying BirA enzyme includes the following steps: lysing the engineered bacterial cells induced by the above method, collecting the supernatant containing BirA enzyme; and purifying the supernatant by nickel column affinity chromatography.
[0017] A BirA enzyme purified by the above method, wherein the C-terminus of the BirA enzyme is fused with an 8×histidine tag and its purity is higher than 90%.
[0018] Application of the above-mentioned BirA enzyme in catalyzing the biotinylation labeling of proteins containing AviTag.
[0019] The beneficial effects of this invention are as follows: 1. This invention uses a prokaryotic expression system (pET-28A vector and BL21(DE3) strain), which has simple culture conditions, short cycle, and low cost, and is suitable for large-scale preparation. By introducing an 8×histidine tag (8×His) at the C-terminus of the BirA enzyme gene, the expression product can be purified in one step by high affinity chromatography (High Affinity Ni-Charged Resin FF Prepacked Column), which is simple and has high purification efficiency.
[0020] 2. This invention effectively reduces the formation of inclusion bodies and promotes the soluble expression of the target protein by specifically optimizing the induction expression conditions (18℃ for 6 hours). Experimental results show that this condition reduces inclusion bodies while maintaining an expression yield similar to that at 37℃, which is beneficial for maintaining the native conformation and activity of the enzyme.
[0021] 3. The purified BirA enzyme of this invention was tested and found to have a molecular weight consistent with the theoretical value (36kD) and high purity, ensuring the accuracy and consistency of subsequent applications. The purified BirA enzyme showed high catalytic efficiency in activity testing, and could catalyze the biotinylation labeling of about 90% of AviTag-containing substrate proteins, proving that the enzyme prepared by the method of this invention has excellent biological functions and can be directly used for subsequent protein labeling and other studies. Attached Figure Description
[0022] Figure 1 This is a schematic diagram of the pET-28A-pET-28A-BirA plasmid in this invention; Figure 2 This is a schematic diagram of PCR in the amplification of the target fragment of the BIRA enzyme and plasmid construction in this invention. Figure 3 This is a schematic diagram illustrating the optimal expression conditions for the soluble BirA enzyme in this invention. Figure 4 This is a schematic diagram illustrating the purification and purity detection of soluble BirA enzyme in this invention. Figure 5 This is a schematic diagram illustrating the verification of BirA enzyme activity in this invention. Detailed Implementation
[0023] The technical solution of the present invention will be further described in detail below with reference to specific embodiments.
[0024] Example 1: Prokaryotic expression and purification of BirA enzyme I. Plasmid: pET-28A II. Experimental Reagents
[0025] III. Experimental Consumables
[0026] IV. Primer Sequence Underlined lines represent homologous arms, and wavy lines represent 8*His;
[0027] V. Experimental Equipment and Instruments
[0028] VI. Preparation of Experimental Reagents LB medium (1L):
[0029] VII. Sequence Information Table
[0030] The underscore is 8*His;
[0031] The underscore is 8*His; VIII. Experimental Methods 6.1 Amplification of the BirA enzyme gene fragment: S1: Add 10 μL of DH5α glycerol bacteria to 5 mL of LB and incubate overnight at 37°C and 200 rpm for activation; S2: The next day, take 2 mL of the overnight activated bacterial culture, centrifuge at 5000g for 1 min, and discard the supernatant; add 2 mL of PBS to wash once, centrifuge again, and discard the supernatant; S3: Add 500 μL of pure water to resuspend the precipitate, and boil at 100℃ for 5 min; S4: Centrifuge at 10000 rpm for 5 min and collect the supernatant; S5: Construct a PCR system and amplify it according to the following conditions;
[0032] S6: Construct the PCR system; S7: Separate the target fragment using 2% agarose gel electrophoresis; S8: Cut off the target band and recover the target fragment using a gel recovery kit.
[0033] 6.2 Acquisition of the linear pET-28A vector: S1: Design reverse primers, obtain linear vectors by reverse PCR amplification, and construct the system under the following conditions;
[0034] S2: Linear carriers were separated by 1% agarose gel electrophoresis; S3: Cut off the target band and recover the linear vector using a gel recovery kit.
[0035] 6.3 Homologous recombination to construct the pET-28A-BirA plasmid: S1: Mix 50 ng of linear vector with 20 ng of gene fragment, adjust the volume to 2.5 μL, add 2.5 μL of homologous recombinase, incubate on ice for 10 min, then transform into BL21(DE3) competent cells. After heat shock transformation, add 1 mL of LB medium and culture for 1 h.
[0036] S2: Spread onto LB agar plates containing kanamycin resistance and incubate overnight.
[0037] S3: The next day, select single colonies and transfer them to LB culture containing kanamycin resistance. Amplify the culture and send the bacterial culture for sequencing to verify the plasmid sequence. Add 50% glycerol at a 1:1 ratio to the remaining bacterial culture and freeze at -20°C.
[0038] 6.4 Protein Expression and Purification S1: Take 10 μL of frozen BL21(DE3) glycerol bacteria containing pET-28A-BirA plasmid and add it to 5 mL of LB culture medium containing kanamycin resistance. Incubate overnight at 37°C and 200 rpm for activation.
[0039] S2: The next day, expand the culture from 5 mL of overnight activated bacterial solution to 500 mL. When OD600 = 0.6-0.8, add IPTG to a final concentration of 0.5 mM to induce protein expression, and continue culturing for several hours.
[0040] S3: Centrifuge at 5000g for 10 minutes, discard the supernatant, and collect the bacterial sludge.
[0041] S4: Add 10 mL of PBS to resuspend the bacterial sludge and lyse the bacterial cells by ultrasonication.
[0042] S5: Centrifuge at 10000g for 10 minutes and collect the supernatant.
[0043] S6: The culture supernatant after centrifugation was purified using a High Affinity Ni-Charged Resin FF Prepacked Column.
[0044] S7: Collect the finally purified protein and verify its molecular weight and purity.
[0045] 6.5 BirA enzyme activity assay.
[0046] IX. Experimental Results 1. Schematic diagram of pET-28A-pET-28A-BirA plasmid like Figure 1 As shown, firstly, the BirA enzyme (U00096.3) gene fragment was queried through the GenBlank database. Its length is 963bp and the protein molecular weight is 36kD. Considering the subsequent purification needs, an 8His tag was designed at the C end of the BirA enzyme for purification.
[0047] 2. Amplification of the target fragment of BIRA enzyme and construction of plasmid like Figure 2As shown, the target fragment was amplified from the genomic DNA of DH5α *Escherichia coli* using PCR. The figure shows the product of amplification of the extracted genomic DNA using the designed F-BirA and R-BirA primers, characterized by 2% agarose gel electrophoresis. A distinct single band of approximately 1000 bp can be observed, consistent with the base count of the target fragment of the BirA enzyme, indicating successful amplification. The target fragment was subsequently purified by gel extraction and ligated to a linear vector via homologous recombination.
[0048] 3. Optimal expression conditions for soluble BirA enzymes were tested. like Figure 3 As shown in the figure, to obtain high-yield soluble expression of BirA enzyme, the soluble expression yields at 37℃ and 18℃ were first tested. The figure shows that expression induced at 37℃ for 5 hours and at 18℃ for 6 hours yielded similar soluble expression yields. However, 18℃ significantly reduced the production of inclusion body proteins. Therefore, induced expression at 18℃ is a suitable condition, which also helps to reduce the impact of high temperature on enzyme activity.
[0049] 4. Purification and purity determination of soluble BirA enzyme like Figure 4 As shown, the induced E. coli were disrupted by sonication, and the collected supernatant was purified using a HighAffinity Ni-Charged Resin FF Prepacked Column. It can be seen that the His-tagged BirA enzyme was successfully purified from the supernatant. The molecular weight of the purified product is consistent with the theoretical value (36 kDa) and exhibits high purity.
[0050] 5. BirA enzyme activity verification like Figure 5 As shown, purified BirA enzyme was mixed with Avitag-containing proteins. In the presence of ATP, magnesium ions, and biotin, BirA enzyme catalyzed the specific covalent coupling of biotin to lysine residues on Avitag. After enzymatic reaction, the Avitag protein was co-incubated with streptavidin, and the activity of BirA enzyme was evaluated by detecting gel migration rate. This is because the biotinylated protein forms a large molecular complex with streptavidin, resulting in a slower migration rate, while the migration rate of the unbiotinylated protein remains unchanged. The results show that the BirA enzyme prepared by this method has high activity, achieving biotinylation labeling in nearly 90% of Avitag-containing proteins after enzymatic reaction.
[0051] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.
Claims
1. A recombinant expression vector for expressing BirA enzyme, characterized in that, The recombinant expression vector is a pET-28A-BirA plasmid constructed by inserting the BirA enzyme-encoding gene into the multiple cloning site of the pET-28A vector; the C-terminus of the BirA enzyme-encoding gene is fused with a nucleotide sequence encoding an 8×histidine tag.
2. The method for expressing BirA enzyme in prokaryotic Escherichia coli and purifying it according to claim 1, characterized in that, The nucleotide sequence of the BirA enzyme encoding gene is SEQ ID NO:
1.
3. A recombinant Escherichia coli engineered bacterium expressing BirA enzyme, characterized in that, The engineered bacteria comprise the recombinant expression vector as described in claim 1 or 2.
4. The recombinant Escherichia coli engineered strain according to claim 3, characterized in that, The host bacterium of the engineered bacteria is Escherichia coli BL21(DE3).
5. A method for recombinant expression of BirA enzyme, characterized in that, The method includes the steps of culturing the recombinant Escherichia coli engineered strain as described in claim 3 and inducing the expression of the BirA enzyme.
6. The recombinant expression method according to claim 5, characterized in that, The induced expression was achieved using IPTG as the inducer; the final concentration of IPTG for induction was 0.5 mM.
7. The recombinant expression method according to claim 5, characterized in that, The induction temperature was 18℃, and the induction time was 5-6 hours.
8. A method for purifying BirA enzyme, characterized in that, The method includes the following steps: lysing the engineered bacterial cells induced by the method of claim 5, collecting the supernatant containing BirA enzyme; and purifying the supernatant by nickel column affinity chromatography.
9. A BirA enzyme purified by the method of claim 8, characterized in that, The BirA enzyme has an 8×histidine tag fused to its C-terminus, and its purity is higher than 90%.
10. The use of the BirA enzyme of claim 9 in catalyzing the biotinylation labeling of proteins containing AviTag.