A fusion tag peptide for efficiently expressing and purifying recombinant proteins in Escherichia coli host cells and its application

Abstract

The present invention provides a fusion tag peptide for efficiently expressing and purifying recombinant proteins in Escherichia coli host cells and its application, belonging to the field of genetic engineering and molecular biology. The fusion tag peptide designed by the present invention comprises a domain T7tag that is conducive to initiating gene transcription level, two affinity purification tag domains 6×His tag and Strep II tag, and a protease recognition domain EK site, and is fused to the N-terminus of the target recombinant protein. The present invention can effectively improve the expression level of recombinant protein, realize efficient expression and purification of target recombinant protein, and has good universality and application scenarios.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering and molecular biology, and specifically relates to a fusion tag peptide for efficiently expressing and purifying recombinant proteins in Escherichia coli host cells and an application thereof. Background Art

[0002] Escherichia coli has attracted much attention due to its advantages such as clear genetic background, simple technical operation and culture conditions, and large-scale fermentation economy. At the same time, Escherichia coli is also the most widely used expression system at present. However, this system also has certain limitations, mainly including the low expression level of some recombinant proteins, possible inclusion body expression due to incorrect folding, unstable and easy degradation of proteins, etc. Not every gene can be effectively expressed in it, which increases the difficulty of preparing and purifying the target protein. Therefore, the invention and application of fusion tags effectively solve the problems related to the expression of recombinant proteins in Escherichia coli. Fusion tag technology is a genetic recombination technology that emerged in the late 20th century. Its main process is to use recombinant DNA technology to fuse a certain tag coding gene at the 3' end or 5' end of the target protein coding gene, express the recombinant protein through a suitable host, and the expressed recombinant protein can be purified by binding the fused tag to the specific ligand coated on the solid phase matrix. Its application not only facilitates the separation and purification of recombinant proteins, but also has the functions of promoting protein expression, mediating correct protein folding, and increasing protein stability.

[0003] A series of common fusion tags have been developed, including 6×His tag, Strep tag, small molecule ubiquitin-like modifier protein (SUMO), glutathione transferase (GST), maltose binding protein (MBP), transcription termination anti-termination factor (NusA), etc. However, their functions are relatively simple and they all have their limitations. They are generally only helpful for affinity chromatography purification or detection and identification of target proteins. One purification tag can only be used for one affinity purification, and relatively large fusion tags affect the function of recombinant proteins. There is no universal fusion tag that can solve the problem of high expression of most proteins and obtaining high-purity recombinant proteins. Therefore, providing a fusion tag peptide with strong universality and high expression level is of great significance for optimizing and improving the recombinant protein genetic engineering and molecular biology technology system based on Escherichia coli. Summary of the invention

[0004] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a fusion tag peptide for efficiently expressing and purifying recombinant proteins in Escherichia coli host cells and its application, and to effectively improve the expression level of recombinant proteins and achieve efficient purification of target proteins by designing fusion tag peptide combination sequences.

[0005] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0006] The present invention provides a fusion tag peptide for efficiently expressing and purifying recombinant proteins in Escherichia coli host cells, comprising a T7 tag domain that is beneficial to initiating gene transcription, two affinity purification tag domains, a 6×His tag and a Strep II tag, and an EK site of a protease recognition domain; the amino acid sequence of the fusion tag peptide is shown in SEQ ID NO: 1.

[0007] The functional domain of the fusion tag peptide designed by the present invention is as follows Figure 1 As shown, the secondary structure prediction of the fusion tag peptide transcribed RNA sequence is as follows Figure 2 shown.

[0008] Preferably, the fusion tag peptide is fused to the N-terminus of the target recombinant protein.

[0009] The present invention provides a nucleic acid encoding the fusion tag peptide, and the sequence of the nucleic acid is shown in SEQ ID NO:2.

[0010] The invention provides a recombinant vector, comprising an expression vector, wherein a nucleic acid encoding a fusion tag peptide is inserted into the expression vector.

[0011] Preferably, the expression vector is an Escherichia coli pET24a expression vector.

[0012] The invention provides a method for constructing the recombinant vector, wherein the fusion tag peptide is cloned between the NdeI and BamHI restriction sites of the expression vector pET24a to construct the recombinant vector.

[0013] The present invention provides a method for improving the expression amount of a target protein by utilizing a fusion tag peptide, comprising the following steps: constructing the above-mentioned recombinant vector, cloning a gene fragment after codon optimization of the target protein into the recombinant vector, wherein the cloning sites are preferably BamHI and EcoRI, and then transforming the recombinant vector carrying the gene fragment of the target protein into an expression host, and then inducing expression.

[0014] Preferably, the expression host is Escherichia coli BL21 (DE3) pLysS strain.

[0015] The present invention provides application of the fusion tag peptide in the industrial production of protein drugs, recombinant cytokines, recombinant collagen, recombinant protein antigens and recombinant protein vaccines.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The fusion tag peptide designed by the present invention is short in length, and the nucleic acid sequence has no stable higher-order structure. It not only significantly improves the expression of the recombinant protein, but also has two high-affinity functional domains, which can bind to specific affinity resins or affinity ligands, thereby achieving efficient purification of the target protein. The end of the fusion tag peptide carries a specific protease recognition site, which can be efficiently removed by a specific protease to obtain a tag-free recombinant protein. Compared with the prior art, the fusion tag peptide provided by the present invention has strong universality, and compared with the ordinary His tag, the protein expression is 4 times higher, and the protein purity is increased by 40%, which has great market prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a diagram of the functional domain structure of the fusion tag peptide.

[0019] Figure 2 This is the predicted secondary structure of the RNA sequence transcribed by the fusion tag peptide.

[0020] Figure 3 The expression of B2UM07 protein was amplified by 1L (the control group did not add the fusion tag of the present invention); Lane A: protein expression of the experimental group, Lane B: protein expression of the control group.

[0021] Figure 4 The expression of ALDH2 protein amplified 1L (the control group did not add the fusion tag of the present invention); Lane A: protein expression of the experimental group, Lane B: protein expression of the control group.

[0022] Figure 5 The expression of Nb-211 protein amplified 1L (the control group did not add the fusion tag of the present invention); Lane A: protein expression of the experimental group, Lane B: protein expression of the control group. DETAILED DESCRIPTION

[0023] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0024] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0025] Materials and reagents used in the following examples:

[0026] Bacterial strain: E. coli BL21 (DE3) pLysS protein expression host bacteria was purchased from Weidi Company.

[0027] LB liquid medium, LB solid medium, 100 mg / mL ampicillin, prepared by our company (Hongxun Biotechnology).

[0028] 1M isopropylthio-β-D-galactoside (IPTG): Weigh 2.31 g IPTG, dissolve it in 6 mL ultrapure water, dilute to 10 mL, and then filter it with a 0.22 μm filter membrane and store it at -20°C. IPTG was purchased from Sinopharm Reagent Co., Ltd. 0.22 μm filter membrane was purchased from Minisart High Flal Co., Ltd.

[0029] 1M DTT: Weigh 1.543 g DTT, dissolve in 6 mL ddH2O, dilute to 10 mL, and store at -20°C. DTT was purchased from BBI.

[0030] 5×SDS-PAGE loading buffer: 12.5mL of 1M Tris-HCl buffer (pH6.8), 25mL of glycerol, 5g of SDS, 0.25g of bromophenol blue, dissolve with ddH2O, dilute to 80mL, and store in a refrigerator at 4℃. Each time you use it, take 2mL of the above solution and 0.5mL of 1M DTT in a ratio of 4:1, mix thoroughly before use, and store in a refrigerator at 4℃.

[0031] 5×Tris-Gly electrophoresis buffer: weigh 15.1 g Tris base, 94 g glycine, and 5 g SDS, stir and dissolve with about 800 mL ddH20, then make up to 1 L and store at room temperature.

[0032] SDS-PAGE staining solution: prepare the solution in the ratio of anhydrous ethanol: ddH2O: glacial acetic acid = 4.5:4.5:1, add 1g Coomassie Brilliant Blue R250, dissolve it fully, then make up to 1L and store it at room temperature.

[0033] Decolorizing solution I: Prepare the solution in the ratio of anhydrous ethanol: ddH2O: glacial acetic acid = 25:65:8 and store at room temperature.

[0034] Decolorizing solution II: Prepare the solution in the ratio of anhydrous ethanol: ddH2O: glacial acetic acid = 10:75:15 and store at room temperature.

[0035] Example 1: Synthesis of fusion tag peptide gene

[0036] The fusion tag peptide with an amino acid sequence as shown in SEQ ID NO: 1 was designed. The company prepared the fusion tag peptide gene by gene synthesis. The nucleotide sequence of the fusion tag peptide after codon optimization was shown in SEQ ID NO: 2.

[0037] Amino acid sequence of fusion tag peptide (SEQ ID NO: 1):

[0038] MASMTGGQQMGRGSHHHHHHWSHPQFEKVGTGSNDDDDK

[0039] Nucleotide sequence of fusion tag peptide (SEQ ID NO: 2):

[0040] atggcgagcatgaccggcggccagcagatgggccgtggttctcatcatcatcaccatcattggagccacccgcag tttgaaaaagtgggcaccggcagcaacgatgatgatgatgataaa

[0041] Example 2: Construction of expression vectors for the experimental group and the control group using B2UM07 as the target recombinant protein gene

[0042] 1. Construction of expression vectors in the experimental group

[0043] Nucleotide sequence of B2UM07 (SEQ ID NO: 4):

[0044]

[0045] The fusion tag peptide gene synthesized in Example 1 was cloned between the NdeI and BamHI restriction sites of the expression vector pET24a to form a recombinant vector, and the nucleotide sequence of the recombinant vector is shown in SEQ ID NO: 3; the gene fragment (SEQ ID NO: 4) after codon optimization of B2UM07 was cloned into the above recombinant vector through the BamHI and EcoRI restriction sites to form the expression vector of the experimental group.

[0046] 2. Construction of expression vector for control group

[0047] The codon-optimized gene fragment of B2UM07 (SEQ ID NO: 4) was cloned between the NdeI and XhoI restriction sites of the expression vector pET24a, the stop codon of the target gene was removed, and the stop codon on the vector was used to terminate the translation to form a control expression vector.

[0048] Example 3: Construction of expression vectors for the experimental group and the control group using ALDH2 as the target recombinant protein gene

[0049] Nucleotide sequence of ALDH2 (SEQ ID NO:5):

[0050]

[0051] The gene fragment (SEQ ID NO: 5) after ALDH2 codon optimization was used as the target recombinant protein gene to construct the experimental group expression vector and the control group expression vector. The construction method was referred to Example 2.

[0052] Example 4: Construction of expression vectors for the experimental group and the control group using Nb-211 as the target recombinant protein gene

[0053] Nucleotide sequence of Nb-211 (SEQ ID NO:6):

[0054] tgcggcggcgaagaaccgcagaacgcgaaacgcaaactgattgtggcgaccgatgcgaccctgccgccgatgagctttctgaacgatcagaacc gcttagcgggctttgaagtggatctgattggcgcggtggcgcgcgaagcgggttttgaatatgacctgattaacgtggaatggaacggcctgtt tggcggcctgattaccaagaagtatgatctggtgattagcagcgtgaccattctggaagaacgcaaagaacgcatggcgtttagcgtgccgtat ctgcagagcggcctgagcctggtggttcgtcgcgatactgaaggcgtgaccagcttagaagatgtgcaggcgcaggatggcgttgttggtgcgc aacgtgcaaccaccgcgttcttttatctggaagattatccggaactgaacaaacaggcgtatgaactgtatggccatgcgatccaggatctgat taaaggcgaaattaccgcggtgctgggcgaaagcaccggcaccctgtattataaaaacaacgatgcggcggtgtttcgcgaaattaagatggtg ggcgatattctgaccgaagaacattatggcattgtggcgcgcaaaggcgaaaccgaactgctgcagcgcgtgaacgatgcgctgaagaaactgc tggatgatggcaccgtgcagcgcctgcatgaaaaatgggaactgggccaggcggcgatggtgccgaaaaccgtggcgtctggtaaagaaaactaa

[0055] The gene fragment (SEQ ID NO: 6) after Nb-211 codon optimization was used as the target recombinant protein gene to construct the experimental group expression vector and the control group expression vector. The construction method was referred to Example 2.

[0056] Example 5: Plasmid transformation and induced expression

[0057] 1. Take 1 μL of the expression vector plasmids of the experimental group and the control group in Example 2-4 and add them to 100 μL of BL21 (DE3) pLysS competent bacteria, and place them on ice for 20 minutes;

[0058] 2. Heat shock at 42℃ for 90 seconds, then quickly place on ice for 3 minutes; add 500μL LB culture medium;

[0059] 3. Incubate at 37°C, 220 rpm with shaking for 1 h, spread 200 μL of bacterial solution on LB plate containing 50 μg / mL Kan, and invert and incubate at 37°C overnight;

[0060] 4. The next morning, pick two BL21(DE3)pLysS clones from each group and inoculate them into 4 mL LB culture medium containing 50 μg / mL Kan in a test tube. Cultivate at 37°C and 220 rpm with shaking until the OD is about 1.5. Take 500 μL of the culture medium to preserve the strain, and inoculate the rest into 1 L fermentation medium.

[0061] 5. Inoculate 1L of fermentation medium in the previous step, shake and culture at 37°C and 220rpm until the OD is about 0.5, add inducer IPTG to a final concentration of 1mM, and culture overnight at 220rpm and 16°C;

[0062] 6. Centrifuge at 6000 rpm for 5 min to remove the supernatant, collect the fermented bacteria, and crush and purify them;

[0063] 7. For capture purification, nickel column affinity chromatography was used, and 20 mM Tris, 500 mM NaCl (pH = 7.4) was used as the equilibrium buffer. The target protein was present in the imidazole eluent;

[0064] 8. Fine purification: Use Strep II tag affinity filler for affinity chromatography, 20mM Tris, 500mM NaCl (pH=7.4) as equilibrium buffer, and thiobiotin to elute the target protein;

[0065] 9. SDS-PAGE analysis of the purified recombinant protein products showed that the protein sizes of Examples 2-4 were approximately 88.22 kDa, 60.67 kDa, and 32.07 kDa, respectively, which was consistent with expectations. Compared with the recombinant protein with only the His tag, the expression level of the target recombinant protein with the fusion tag of the present invention and the purity of the obtained product were significantly improved. The results of SDS-PAGE are shown in Figure 3 , Figure 4 , Figure 5 .

[0066] Example 6: Universality experiment of fusion tag peptide

[0067] In order to prove the universal adaptability of the fusion tag peptide designed by the present invention, B2UM07, ALDH2, and Nb-211 were selected as genes to be expressed for comparative experiments. These three genes had no expression when expressed alone, and had low expression levels when combined with other tags. After the N-terminal fusion tag peptide in Example 1 of the present invention was applied, the experimental results were as follows: Figure 3-Figure 5 As shown, the details are as follows:

[0068] B2UM07 protein expression: The expression level of B2UM07 protein using the fusion tag peptide of the present invention is about 4 times higher than that of the ordinary His tag, and the protein purity is increased by about 40%.

[0069] ALDH2 protein expression: The expression level of ALDH2 protein using the fusion tag peptide of the present invention is about 2 times higher than that of the common His tag, and the protein purity is increased by about 10%.

[0070] Expression of Nb-211 protein: The expression level of Nb-211 protein using the fusion tag peptide of the present invention is about 2.4 times higher than that of the common His tag, and the protein purity is increased by about 20%.

[0071] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. Application of a fusion tag peptide for efficient expression and purification of recombinant proteins in Escherichia coli host cells in the industrial production of B2UM07 protein, ALDH2 protein or Nb-211 protein, It is characterized in that The fusion tag peptide comprises a T7 tag domain that is beneficial to initiating gene transcription, two affinity purification tag domains, a 6×His tag and a Strep II tag, and an EK site of a protease recognition domain; the amino acid sequence of the fusion tag peptide is shown in SEQ ID NO: 1, and the fusion tag peptide is fused to the N-terminus of the target recombinant protein; The B2UM07 protein is encoded by the nucleotide sequence shown in SEQ ID NO:4, the ALDH2 protein is encoded by the nucleotide sequence shown in SEQ ID NO:5, and the Nb-211 protein is encoded by the nucleotide sequence shown in SEQ ID NO:6.

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