Application of fusion tag NusA in escherichia coli expression PLCG2 protein

Abstract

The invention relates to a method for promoting soluble expression of a protein in escherichia coli by using a protein fusion tag, which is characterized by comprising the following steps of: adding a segment of sequence of the fusion tag on an existing expression vector by using a fragment amplification and enzyme digestion and enzyme ligation method; and cloning the sequence of the target protein onto the modified expression vector, transforming the correctly constructed expression vector into escherichia coli for induced expression, and detecting the expression condition and the expression quantity by using SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis). The problem that the soluble expression amount of the PLCG2 protein in escherichia coli is small is solved, tedious steps in the protein purification process are reduced, a better immunogen is provided for an animal immune test with the protein in the next step, and the detection accuracy and precision of a kit with the protein and the antibody as the core are further improved.

Description

Technical Field

[0001] The present invention relates to a method for promoting the soluble expression of a protein in Escherichia coli by using a protein fusion tag. Background Art

[0002] Phosphatidylinositol-4,5-bisphosphate (PIP2) is a phospholipid. It accounts for less than 1% of membrane phospholipids, yet its functions are highly complex. This low-abundance polyphosphoinositide lipid can be hydrolyzed by phospholipase C, activated by hormone or cytokine receptors, into the second messenger molecules inositol triphosphate (IP3) and diacylglycerol (DAG), which participate in signal transduction.

[0003] Phosphodiesterases (PDEs) hydrolyze the intracellular second messengers cyclic adenosine monophosphate (cAMP) or cyclic guanosine monophosphate (cGMP), degrading them and terminating the biochemical effects these second messengers transmit. cAMP and cGMP play important regulatory roles in cellular activity, and their concentrations are primarily determined by the balance between synthesis by adenylate cyclase and hydrolysis by phosphodiesterases (PDEs). PDEs are widely distributed in the human body, and their physiological effects span multiple research areas. In recent years, PDEs have attracted widespread attention as novel therapeutic targets, becoming a new research hotspot. Immunological methods are undoubtedly the most convenient way to detect PEDs proteins. Immunological detection requires the development of a test method and the preparation of a kit. The core component of the kit—the antibody—is traditionally produced using recombinant proteins. The general process involves constructing an expression strain based on the properties and intended use of the target protein, enabling it to express the desired protein. The strain is then cultured and induced for expression. The bacteria are then harvested and lysed to release the target protein. The lysate is then purified using various methods depending on the target protein's fusion tag and expression pattern. The purified target protein is then used for animal immunization to generate polyclonal antibodies, which are then purified. This protocol is cumbersome, and the cost of proteases is high, making it unsuitable for large-scale industrial production. Furthermore, each additional step results in a loss of at least 20% protein and approximately 30% immunological activity.

[0004] If a recombinant protein is expressed as inclusion bodies (incompletely folded peptide chains with altered structure and function), not only will the purification process be more complex and difficult, but the activity and quality of the protein cannot be guaranteed, directly impacting subsequent experimental research. Because foreign proteins, especially human proteins, are easily formed into inclusion bodies when expressed in E. coli due to the lack of a post-translational modification system, expression may be impossible or even at low levels. To address this issue, various protein tags have emerged.

[0005] NusA is a native Escherichia coli protein, a transcription antitermination factor, composed of approximately 496 amino acids and with a molecular weight of approximately 55 kDa. It was screened by Davia in 1999 from a library of 4,000 E. coli proteins. Research results have shown that most proteins containing the NusA tag are expressed in soluble form, while proteins expressed alone or in fusion with a thioredoxin tag (Trx-tag, a fusion tag commonly used to enhance solubility) express as inclusion bodies. NusA, a highly soluble protein tag, can significantly enhance the solubility of recombinant proteins when fused to them. Summary of the Invention

[0006] The purpose of the present invention is to overcome the problems existing in the above-mentioned prior art and to provide an effective and practical method for promoting protein expression in a soluble form during protein expression, thereby simplifying the subsequent purification steps and effectively improving the quality of the protein product.

[0007] NusA is a native Escherichia coli protein, a transcription antitermination factor, composed of approximately 496 amino acids and a molecular weight of approximately 55 kDa. Research results indicate that most proteins containing the NusA tag are expressed as soluble proteins, while proteins expressed alone or in fusion with a thioredoxin tag (Trx-tag, a fusion tag commonly used to enhance solubility) express as inclusion bodies. NusA, a highly soluble protein tag, can significantly enhance the solubility of recombinant proteins when fused to them.

[0008] The technical solution of the present invention is: A method for promoting the soluble expression of a protein in Escherichia coli using a protein fusion tag is characterized by the following steps: adding a fusion tag sequence to an existing expression vector by fragment amplification and enzyme digestion and ligation, then cloning the sequence of the target protein into the modified expression vector, transforming the correctly constructed expression vector into an expression host for induced expression, and detecting the expression status and expression level of the target protein by SDS-PAGE.

[0009] The protein fusion tag is a NusA protein tag, the target protein is a PLCG2 protein, the expression vector is pET28a, the cloning bacteria is Escherichia coli DH5α, the expression host is Escherichia coli Rosetta and Escherichia coli C41 strains, and the pET series vectors commonly used in the expression system in Escherichia coli also include pET30a and pET32a. This method is not limited to one vector.

[0010] The PLCG2 protein of the present invention can be expressed in large quantities, and the soluble expression reaches more than 95%.

[0011] The construction of an expression strain for soluble expression of PLCG2 protein was carried out in the following steps: 1) Search the sequence of NusA at NCBI (National Center for Biotechnology Information), remove the terminal stop codon, design primers with restriction sites NdeⅠ and XhoⅠ, perform PCR amplification, and identify by agarose gel electrophoresis. If no nonspecific bands are found, recover the NusA fragment using a cleanup kit; if nonspecific bands are found, recover the fragment using a gel recovery kit. 2) The recovered NusA fragment and vector pET28a were double-digested with NdeⅠ and XhoⅠ, respectively, and detected by agarose gel electrophoresis. The NusA fragment with double-digestion sites NdeⅠ and XhoⅠ was recovered using a clean recovery kit, and the linear vector pET28a with double-digestion sites NdeⅠ and XhoⅠ was recovered using a gel recovery kit; 3) The digested products were recovered and ligated with T4 ligase, and transformed into Escherichia coli DH5α. The correct transformants were screened based on their resistance, and then identified by PCR and sequencing. 4) Identify the correct transformants, expand the culture, and extract the plasmid NusA-pET28a; 5) Search the plcg2 sequence information at NCBI, analyze the protein properties, select the appropriate stage fragment according to the requirements of the immunogen, perform codon optimization, and synthesize the entire gene at Wuhan Tianyi Huiyuan Biotechnology Co., Ltd. and clone it into the pET28a vector; 6) Based on the optimized plcg2 sequence information, primers with restriction enzyme cleavage sites NcoⅠ and NdeⅠ were designed for PCR amplification and identification by agarose gel electrophoresis. If no nonspecific bands were found, the plcg2 fragment was recovered using a clean recovery kit; if nonspecific bands were found, the fragment was recovered using a gel recovery kit; 7) The recovered plcg2 fragment and vector NusA-pET28a were double-digested with NcoⅠ and NdeⅠ, respectively, and detected by agarose gel electrophoresis. The NusA fragment with double-digestion sites NcoⅠ and NdeⅠ was recovered using a clean recovery kit, and the linear vector NusA-pET28a with double-digestion sites NcoⅠ and NdeⅠ was recovered using a gel recovery kit; 8) The digested products were recovered and ligated with T4 ligase, and transformed into Escherichia coli DH5α. The correct transformants were screened based on resistance, and identified by PCR and sequencing.

[0012] The principle underlying the present invention is that NusA is a protein of E. coli itself. Fusion expression of proteins that cannot or only express in small amounts in soluble form in E. coli with it can promote their conversion from inclusion bodies to soluble forms, thereby improving the structure and properties of the protein, simplifying subsequent purification work, and improving the quality of antibodies obtained in animal immunization tests. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 and Figure 2 To construct vector plcg2 - NusA - Schematic diagram of pET28a.

[0014] Figure 3 For the present invention NusA Amplification of fragments.

[0015] Figure 4 For the present invention plcg2 Amplification of fragments.

[0016] Figure 5 For the present invention NusA -PCR verification of pET28a-DH5α transformants.

[0017] Figure 6 For the present invention plcg2 - NusA -PCR verification of pET28a-DH5α transformants.

[0018] Figure 7 The expression of PLCG2 protein.

[0019] Figure 8 PCR system for amplifying NusA tag sequences. DETAILED DESCRIPTION

[0020] 1. Materials and Reagents 1.1 Strains and plasmids E. coli DH5α and E. coli Rosetta strains are preserved by our company; 1.2 Reagents and kits Restriction enzymes Nco I, Nde I, Xho I, and T4 ligase were purchased from TaKaRa; Plasmid Mini Kit, Gel Extraction Kit, and Cycle Pure Kit were purchased from OMEGA Biotechnology Co., Ltd. 1.3 Culture medium LB medium: 10 g / L yeast extract, 20 g / L peptone, 10 g / L NaCl. For solid medium, add 15 g / L agar. Sterilize at 121°C for 30 min.

[0021] 1.4 Solution 50 mg / mL kanamycin: Weigh 3 g of kanamycin powder, dissolve in distilled water, and dilute to 100 mL. Filter through a 0.22 μm sterile filter membrane and aliquot into sterile 1.5 mL centrifuge tubes and store at -20°C. 1× PBS buffer (pH 7.5): 132 mL 1 mol / L K2HPO4, 868 mL 1 mol / L KH2PO4, adjust the pH to 7.5 ± 0.1 with phosphoric acid or KOH; 2× loading buffer: 1.25 mL 0.5 mol / L Tris-HCl (pH 6.8), 0.2 mL 10% SDS, 0.2 mL 0.5% bromophenol blue, 2.5 mL glycerol, dissolve in distilled water and dilute to 10 mL; 30% acrylamide: 29.0 g acrylamide, 1.0 g methylene bisacrylamide, dissolve in distilled water and dilute to 1 L; 1× running buffer: 3.03 g Tris base, 14.4 g glycine, 1.0 g SDS, dissolved in distilled water and adjusted to 1 L; Staining solution: Coomassie Brilliant Blue R-250 1.0 g, methanol 450 mL, glacial acetic acid 100 mL, dissolve in distilled water and dilute to 1 L; Decolorization solution: 100 mL of methanol, 100 mL of glacial acetic acid, dissolve in distilled water and adjust the volume to 1 L.

[0022] 2. Experimental Procedure 2.1 Amplification of NusA tag sequences Based on the NusA gene sequence without the stop codon, primers with NdeⅠ and XhoⅠ restriction sites were designed. PCR reaction was performed using the recombinant plasmid NusA-pUC57 as a template. The reaction conditions were pre-denaturation at 95°C for 5 min, denaturation at 95°C for 1 min 20 s, annealing at 60°C for 30 s, extension at 72°C for 1 min, 34 cycles, and extension at 72°C for 5 min. The PCR system was as follows: reactants volume NusA-pUC57 1 μL NusA-F 1 μL NusA-R 1 μL 2×Mix 15 μL dd H2O 12 μL Total 30 μL .

[0023] 2.2 Construction of the NusA-pET28a vector After the PCR reaction is completed, the PCR product is detected by 1% agarose gel electrophoresis. If the length is consistent with the target gene, the target band is recovered using a cleaning recovery kit, and the concentration and purity are detected using a micro-UV spectrophotometer; The cleaned and recovered NusA fragment and pET28a plasmid were double-digested with NdeⅠ and XhoⅠ, respectively. The enzyme digestion system is as follows: reactants volume 10×Buffer 5 µL NdeⅠ 1 µL XhoⅠ 1 µL DNA 43 µL Total 50 µL .

[0024] After double enzyme digestion, the digestion products were detected by electrophoresis on 1% agarose gel to determine whether the digestion was sufficient, and the gel recovery kit was used to recover them respectively; The digested products were cleaned and recovered and then ligated with T4 ligase. The enzyme ligation system is as follows: reactants volume T4 10×buffer 1 µL T4 ligase 1 µL pET28a 2 µL NusA 6 µL Total 10 µL .

[0025] 2.3 Amplification of plcg2 sequences Based on the optimized plcg2 gene sequence, primers with NcoⅠ and NdeⅠ restriction sites were designed. PCR reaction was performed using the recombinant plasmid plcg2-pET28a as a template. The reaction conditions were pre-denaturation at 95°C for 5 min, denaturation at 95°C for 1 min 20 s, annealing at 60°C for 30 s, extension at 72°C for 1 min, 34 cycles, and extension at 72°C for 5 min. The PCR system was as follows: reactants volume plcg2-pET28a 1 μL plcg2-F 1 μL plcg2-R 1 μL 2×Mix 15 μL dd H2O 12 μL Total 30 μL .

[0026] 2.4 Construction of the plcg2-NusA-pET28a vector After the PCR reaction is completed, the PCR product is detected by 1% agarose gel electrophoresis. If the length is consistent with the target gene, the target band is recovered using a cleaning recovery kit, and the concentration and purity are detected using a micro-UV spectrophotometer; The cleaned and recovered plcg2 fragment and NusA-pET28a plasmid were double-digested with NcoⅠ and NdeⅠ, respectively. The enzyme digestion system is as follows: reactants volume 10×Buffer 5 µL NcoⅠ 1 µL NdeⅠ 1 µL DNA 43 µL Total 50 µL .

[0027] After double enzyme digestion, the digestion products were detected by electrophoresis on 1% agarose gel to determine whether the digestion was sufficient, and the gel recovery kit was used to recover them respectively; The digested products were cleaned and recovered and then ligated with T4 ligase. The enzyme ligation system is as follows: reactants volume T4 10×buffer 1 µL T4 ligase 1 µL NusA-pET28a 2 µL plcg2 6 µL Total 10 µL .

[0028] 2.5 Screening of transformants Remove competent E. coli cells from a -80°C freezer and place on ice for 5 minutes. Add the enzyme-linked product, mix gently, and place on ice for 30 minutes. Heat shock the cells in a 42°C water bath for 90 seconds, then immediately cool on ice for 3-5 minutes. Add 800 µL of LB medium to the centrifuge tube and incubate at 37°C, 200 rpm, for 45-60 minutes. Spread an appropriate amount of the bacterial solution onto an LB plate containing 30 µg / mL Kan. Once the bacterial solution is completely absorbed, incubate the plate in an inverted manner at 37°C for 16-24 hours. After a single colony grows on the antibiotic-added LB plate, pick a single colony and place it in a 1.5 mL sterile centrifuge tube containing 1 mL of LB medium (containing 30 µg / mL Kan). Pick about 5 transformants from each plate and culture at 37°C and 200 rpm for about 6 h. PCR reaction was performed using bacterial suspension as template; PCR products were detected by 1% agarose gel electrophoresis, and positive clones were preliminarily screened based on fragment size; correct transformants were verified and sequenced for further verification; correct transformants were cultured in PA bottles to the appropriate concentration and stored at -80°C with a final glycerol concentration of 25%; 2.6 Inducible expression Use a plasmid extraction kit to extract a small amount of plcg2-NusA-pET28a and plcg2-pET28a, and transform them into expression bacteria Escherichia coli BL21 (DE3) and Escherichia coli C41, respectively. The transformation method is the same as 2.5; Select suitable transformants and transfer them to PA flasks (containing 10 mL of LB medium containing 50 µg / mL kanamycin. For E. coli BL21 (DE3), chloramphenicol should also be added to achieve a final concentration of 50 µg / mL). Grow at 37°C and 180 rpm for 5–6 h until the OD600 reaches approximately 0.5. Take 1 mL of the preserved strain (final glycerol concentration 25%); take 1 mL into a 1.5 mL sterile centrifuge tube as a blank control (uninduced control); take 4 mL into another sterile PA bottle; add 4 μL of sterile IPTG solution (prepared concentration of 238 mg / mL) to each PA bottle. Divide the PA bottle into two and induce them overnight at 16°C and 37°C respectively. The blank control is cultured overnight at 37°C. 2.7 SDS-PAGE detection 2.7.1 Preparation of SDS-PAGE gel Prepare SDS-PAGE gels as shown in the table below. Prepare the separating gel first, and then prepare the stacking gel after it has solidified. .

[0029] 2.7.2 Sample preparation After induction, the PA bottles were removed and 1 mL of bacterial solution was taken from each PA bottle into a 1.5 mL centrifuge tube. The tubes were centrifuged at 6000 rpm for 1 min and the supernatant was discarded. The precipitate was fully resuspended in 300 μL 1× PBS, ultrasonically disrupted in an ice-water bath until the bacterial suspension was clear and translucent, and centrifuged at 12,000 rpm for 30 min. The supernatant was removed and transferred to another 1.5 mL centrifuge tube; the precipitate was dissolved with 300 μL of 500 mM urea solution (prepared with 1× PBS); Take 10 μL of the sample treated by the two methods, add 10 μL of 2× loading buffer, and boil in water for 10 min; 2.7.3 Electrophoresis and staining and decolorization After the samples in the boiling water bath have cooled to room temperature, add the samples to the sample wells of the PAGE gel in order. Set the voltage to 80 V for electrophoresis for 15-20 minutes until bromophenol blue is observed. Then set the voltage to 120 V for electrophoresis for about 90 minutes. Pay attention to the position of the bromophenol blue band and stop the electrophoresis when it approaches the bottom. Remove the PAGE gel, add an appropriate amount of staining solution, heat it slightly, place it on a horizontal shaker, and stain for 30 minutes; pour out the staining solution, add an appropriate amount of destaining solution, heat it slightly, place it on a horizontal shaker, and destain for 30 minutes; change the destaining solution twice until the clean bands are visible to the naked eye.

Claims

1. A method for promoting the soluble expression of a protein in Escherichia coli using a protein fusion tag, characterized in that Proceed as follows: add a fusion tag sequence to the existing expression vector by fragment amplification and enzyme digestion and ligation, then clone the sequence of the target protein into the modified expression vector, transform the correctly constructed expression vector into Escherichia coli for induced expression, and use SDS-PAGE to detect the expression status and expression level.

2. The method according to claim 1, characterized in that: The protein fusion tag is NusA, a transcription anti-termination factor.

3. The method according to claim 1 or 2, characterized in that Follow these steps: synthesis NusA Fragments, primers with double enzyme cutting sites were designed for PCR amplification; Will NusA The fragment and vector pET28a were digested separately and then ligated to obtain the modified vector. NusA- pET28a; synthesis plcg2 Fragments, primers with double enzyme cutting sites were designed for PCR amplification; Will plcg2 Fragments and vectors NusA- After pET28a was digested separately, the expression vector was obtained by enzyme ligation. plcg2-NusA- pET28a; The expression vector was transformed into the expression host Escherichia coli BL21 and Escherichia coli C41, and the transformants were selected for culture and induction of expression; The expression product was detected by SDS-PAGE.

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