TEV protease and soluble expression method thereof in escherichia coli

Abstract

The invention discloses TEV protease and a soluble expression method thereof in escherichia coli. The method comprises the following steps: constructing recombinant plasmids, culturing the recombinant plasmids, adding IPTG (isopropyl-beta-d-thiogalactoside) into a culture system for induced culture, and inducing recombinant bacteria to express TEV protease to obtain induced bacteria; re-suspending and crushing the induced thalli, and purifying the thalli through a nickel column; and removing imidazole in the enzyme solution through a desalting column to obtain the high-purity TEV protease.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and in particular relates to TEV protease and a method for soluble expression thereof in Escherichia coli. Background Art

[0002] TEV protease is a commonly used tool enzyme for removing affinity tags on fusion proteins. It has strong site specificity, strictly recognizes the heptapeptide sequence, and the cleavage site is between glutamine and glycine / serine. When expressing proteins, tags such as His, MBP, and GST are often fused to the N-terminus, and the above-mentioned heptapeptide sequence is designed to be added between the tag and the target protein. After TEV protease cleavage, the N-terminus of the target protein has only one additional G / S amino acid residue, which minimizes the impact on the structure and function of the target protein. Therefore, TEV protease is one of the most widely used enzymes in the biological field. Summary of the invention

[0003] Technical problem to be solved: In view of the technical defects in the prior art of expressing TEV protease in E. coli, most of which are in the form of inclusion bodies with low yield, a method for soluble expression of TEV protease in E. coli with high yield is disclosed.

[0004] Technical solution:

[0005] A TEV protease, the nucleotide sequence of the TEV protease is shown in SEQ ID NO: 1:

[0006] ATGCGTGGTAGTGGCGAAAGTCTGTTTAAAGGTCCGCGTGATTATAATCCGATTAGCAGTAGCATTTGCCATCTGACCAATGAAAGTGATGGCCATACCACCAGTCTGTATGGTATTGGCTTTGGCCCGTTTATTATTACCAATAAGCATCTGTTTCGCCGTAATAATGGTACACTGCTGGTGCAGAGTCTGCATGGCGTGTTTAAAGTTAAAGATACCACCACCCTGCAGCAGCATCTGGTGGATGGTCGCGATATGATTATTATTCGTATGCCGAAAGATTTCCCGCCGTTTCCGCAGAAACTGAAATTTCGTGAACCGCAGCGTGAAGAACGTATTTGTCTGGTTACCACCAATTTTCAGACCAAAAGTATGAGCAGTATGGTTAGTGATACCAGTTGTACCTTTCCGAGCAGCGATGGTACATTTTGGAAACATTGGATTCAGACCAAAGATGGCCAGTGCGGTAATCCGCTGGTGAGTACCCGTGATGGCTTTATTGTTGGTATTCATAGCGCCAGCAATTTTACCAATACCAATAATTATTTCGCAAGCGTGCCGAAAAATTTTATGGAACTGCTGACCAATCAGGAAGCACAGCAGTGGGTGAGTGGCTGGCGTCTGAATGCAGATAGCGTGCTGTGGGGTGGTCATAAAGTTTTTATGAATAAGCCGGAAGAACCGTTTCAGCCGGTTAAAGAAGCCACCCAGCTGATGAATGGTAGCGGCAGCTGGAGTCATCCGCAGTTTGAAAAAGGTGGCGGCAGTCATCATCATCATCACCATTAA。

[0007] The amino acid molecule encoding the TEV protease, the amino acid sequence of which is shown in SEQ ID NO:2:

[0008] MRGSGESLFKGPRDYNPISSSICHLTNESDGHTTSLYGIGFGPFIITNKHLFRRNNGTLLVQSLHGVFKVKDTTTLQQHLVDGRDMIIIRMPKDFPPFPQKLKFREPQREERICLVTTNFQTKSMSSMVSD TSCTFPSSDGTFWKHWIQTKDGQCGNPLVSTRDGFIVGIHSASNFTNTNNYFASVPKNFMELLTNQEAQQWVSGWRLNADSVLWGGHKVFMNKPEEPFQPVKEATQLMNGSGSWSHPQFEKGGGSHHHHHH.

[0009] The present application also discloses a method for soluble expression of TEV protease in Escherichia coli, comprising the steps of:

[0010] Step 1: inserting the TEV protease gene sequence shown in SEQ ID NO: 1 between the restriction endonucleases of the PET-32a vector to obtain the recombinant plasmid pET32a-TEV;

[0011] Step 2: transformation: add 1 μl of recombinant plasmid pET32a-TEV to BL21 (DE3) competent cells, place on ice for 30 min, then heat shock at 42°C for 60 s; place on ice for 3 min; then add 500 μl of LB in a clean bench, place on a 37°C shaker at 200 rpm / min for 1 h to activate the bacteria; pour the activation solution into a plate with kanamycin resistance, seal the plate after the liquid is air-dried, and place in a 37°C incubator overnight;

[0012] Step 3: Activation, expansion, and induction: Pick the cultured monoclonal bacteria and add them to LB liquid culture medium containing 100 μg / mL ampicillin for activation overnight; take 5 mL of the activated bacterial solution, inoculate it into 500 mL of LB liquid culture medium containing 100 μg / mL ampicillin at a volume ratio of 1:100, and shake culture at 37°C and 200 rpm / min until the OD600 value = 0.6; when OD600 = 0.6, add 400 μl of 0.5M IPTG, and induce expression at 23°C and 200 rpm / min for 16 hours to obtain the induced bacterial solution;

[0013] Step 4: Resuspend and break: pour the induced bacterial liquid into the collection bottle, balance, centrifuge at 5000rpm / min for 15min, and obtain 2g of bacterial cells after centrifugation; resuspend with breaking buffer; the collected bacterial cells after resuspending are broken by low-temperature ultrasonic until the bacterial cells are clear. After breaking, centrifuge at 4℃ and 12000rpm / min for 30min, collect the supernatant, and filter the collected supernatant through a 0.45um filter membrane;

[0014] Step 5, affinity chromatography: add the filtered supernatant to a nickel chromatography column containing Ni-NTA agarose gel, load the sample after equilibration with a crushing buffer for 5CV, wash the impurities with 8CV crushing buffer, then elute the impurities with 6CV 10% eluent, and finally elute the target protein with a 10CV gradient elution to obtain TEV protease;

[0015] Step 6, SDS-PAGE detection: collect TEV protease and perform SDS-PAGE to detect the purity of the purified protein; Step 7, desalting: add TEV protease to a desalting column, load the sample after balancing with a desalting buffer for 5CV, elute with 1CV of desalting buffer, remove the imidazole in the TEV protease, and obtain the desalted TEV protease;

[0016] Step 8, TEV enzyme activity determination: The concentration of TEV protease after desalting was determined, and the enzyme activity test was performed. W63 containing TEV protease cleavage site and TEV enzyme were mixed at a molar ratio of 1:10, reacted at 4°C overnight, and the enzyme cleavage effect was detected by SDS-PAGE.

[0017] Preferably, the disruption buffer consists of 50 mM Tris pH 8.0 and 300 mM NaCl.

[0018] Preferably, the 10% eluent consists of 50 mM Tris pH 8.0, 300 mM NaCl and 50 mM imidazole.

[0019] Preferably, the desalting buffer consists of 20 mM Tris pH 8.0 and 100 mM NaCl.

[0020] Preferably, the system buffer for the eighth step reaction consists of 20 mM Tris-HCl (pH 8.0), 100 mM NaCl and 1 mM DTT.

[0021] Beneficial effects: The TEV protease gene is truncated and mutated, and a soluble, high-yield TEV protease is obtained through an E. coli expression system. The soluble expression of TEV protease in E. coli can be achieved, and 1L of fermentation bacteria can be purified to obtain 50mg TEV protease, which reduces the cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a schematic diagram of the structure of the expression vector pET32a-TEV of this application;

[0023] Figure 2 This is the chromatogram of the TEV protease protein purifier of this application;

[0024] Figure 3The sample SDS-APGE detection corresponding to the TEV protease of this application, wherein S: supernatant after ultrasonic disruption; P: precipitate after high-speed centrifugation after ultrasonic disruption; FT: flow-through peak;

[0025] Figure 4 For the activity test of EV protease in this application, the control group: W65 protein; TEV: TEV protease; W65+TEV: W65 protein and TEV protease are mixed in a molar ratio of 10:1. DETAILED DESCRIPTION

[0026] The preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are provided only for the purpose of illustration and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0027] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0028] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0029] Example 1

[0030] A TEV protease, the nucleotide sequence of the TEV protease is shown in SEQ ID NO: 1:

[0031] ATGCGTGGTAGTGGCGAAAGTCTGTTTAAAGGTCCGCGTGATTATAATCCGATTAGCAGTAGCATTTGCCATCTGACCAATGAAAGTGATGGCCATACCACCAGTCTGTATGGTATTGGCTTTGGCCCGTTTATTATTACCAATAAGCATCTGTTTCGCCGTAATAATGGTACACTGCTGGTGCAGAGTCTGCATGGCGTGTTTAAAGTTAAAGATACCACCACCCTGCAGCAGCATCTGGTGGATGGTCGCGATATGATTATTATTCGTATGCCGAAAGATTTCCCGCCGTTTCCGCAGAAACTGAAATTTCGTGAACCGCAGCGTGAAGAACGTATTTGTCTGGTTACCACCAATTTTCAGACCAAAAGTATGAGCAGTATGGTTAGTGATACCAGTTGTACCTTTCCGAGCAGCGATGGTACATTTTGGAAACATTGGATTCAGACCAAAGATGGCCAGTGCGGTAATCCGCTGGTGAGTACCCGTGATGGCTTTATTGTTGGTATTCATAGCGCCAGCAATTTTACCAATACCAATAATTATTTCGCAAGCGTGCCGAAAAATTTTATGGAACTGCTGACCAATCAGGAAGCACAGCAGTGGGTGAGTGGCTGGCGTCTGAATGCAGATAGCGTGCTGTGGGGTGGTCATAAAGTTTTTATGAATAAGCCGGAAGAACCGTTTCAGCCGGTTAAAGAAGCCACCCAGCTGATGAATGGTAGCGGCAGCTGGAGTCATCCGCAGTTTGAAAAAGGTGGCGGCAGTCATCATCATCATCACCATTAA。

[0032] An amino acid molecule encoding the TEV protease described in claim 1, the amino acid sequence of which is shown in SEQ ID NO: 2:

[0033] MRGSGESLFKGPRDYNPISSSICHLTNESDGHTTSLYGIGFGPFIITNKHLFRRNNGTLLVQSLHGVFKVKDTTTLQQHLVDGRDMIIIRMPKDFPPFPQKLKFREPQREERICLVTTNFQTKSMSSMVSD TSCTFPSSDGTFWKHWIQTKDGQCGNPLVSTRDGFIVGIHSASNFTNTNNYFASVPKNFMELLTNQEAQQWVSGWRLNADSVLWGGHKVFMNKPEEPFQPVKEATQLMNGSGSWSHPQFEKGGGSHHHHHH.

[0034] Example 2

[0035] A method for soluble expression of TEV protease in Escherichia coli, comprising the following steps:

[0036] The first step: insert the TEV protease gene sequence shown in SEQ ID NO:1 between the restriction endonucleases of the PET-32a vector to obtain the recombinant plasmid pET32a-TEV; the recombinant plasmid is sequenced and verified, and the plasmid is stored at -80°C after the sequencing is correct; the second step, transformation: add 1 μl of the recombinant plasmid pET32a-TEV to the BL21 (DE3) competent medium, place it on ice for 30 minutes, and then heat shock it at 42°C for 60S; then place it on ice for 3 minutes; then add 500 μl LB in the clean bench, place it on a 37°C shaker, 200 rpm / min, 1 hour, and activate the bacteria; pour the activation solution into a plate with kanamycin resistance, seal it after the liquid is air-dried, and place it in a 37°C incubator overnight;

[0037] Step 3: Activation, expansion, and induction: Pick the cultured monoclonal bacteria and add them to LB liquid culture medium containing 100 μg / mL ampicillin for activation overnight; take 5 mL of the activated bacterial solution, inoculate it into 500 mL of LB liquid culture medium containing 100 μg / mL ampicillin at a volume ratio of 1:100, and shake culture at 37°C and 200 rpm / min until the OD600 value = 0.6; when OD600 = 0.6, add 400 μl of 0.5M IPTG, and induce expression at 23°C and 200 rpm / min for 16 hours to obtain the induced bacterial solution;

[0038] Step 4: Resuspend and break: pour the induced bacterial liquid into the collection bottle, balance, centrifuge at 5000rpm / min for 15min, and obtain 2g of bacterial cells after centrifugation; resuspend with breaking buffer; the collected bacterial cells after resuspending are broken by low-temperature ultrasonic until the bacterial cells are clear. After breaking, centrifuge at 4℃ and 12000rpm / min for 30min, collect the supernatant, and filter the collected supernatant through a 0.45um filter membrane;

[0039] Step 5, affinity chromatography: add the filtered supernatant to a nickel chromatography column containing Ni-NTA agarose gel, load the sample after balancing with the crushing buffer for 5CV, wash the impurities with 8CV crushing buffer, then elute the impurities with 6CV 10% eluent, and finally elute the target protein with a 10CV gradient elution to obtain TEV protease; the purification results are shown in Figure 1 As shown;

[0040] Step 6, SDS-PAGE detection: TEV protease was collected and the purity of the purified protein was detected by SDS-PAGE; the protein gel image of the nickel column purification was as follows: Figure 2 As shown, a large amount of target protein can be seen in the elution peak, indicating that it can be soluble expressed in E. coli and a high-purity enzyme solution can be obtained;

[0041] Step 7, desalting: adding TEV protease to the desalting column, equilibrate with desalting buffer for 5CV before loading, eluting with 1CV desalting buffer to remove imidazole in TEV protease to obtain desalted TEV protease;

[0042] Step 8, TEV enzyme activity determination: After desalting, the concentration of TEV protease was determined, and the enzyme activity test was performed. W63 containing TEV protease cleavage site and TEV enzyme were mixed at a molar ratio of 1:10, reacted at 4°C overnight, and the enzyme cleavage effect was detected by SDS-PAGE. The results are as follows Figure 3 shown.

Claims

1. A TEV protease, characterized in that The nucleotide sequence of TEV protease is shown in SEQ ID NO:1: ATGCGTGGTAGTGGCGAAAGTCTGTTTAAAGGTCCGCGTGATTATAATCCGATTAGCAGTAGCATTTGCCATCTGACCAATGAAAGTGATGGCCATACCACCAGTCTGTATGGTATTGGCTTTGGCCCGTTTATTATTACCAATAAGCATCTGTTTCGCCGTAATAATGGTACACTGCTGGTGCAGAGTCTGCATGGCGTGTTTAAAGTTAAAGATACCACCACCCTGCAGCAGCATCTGGTGGATGGTCGCGATATGATTATTATTCGTATGCCGAAAGATTTCCCGCCGTTTCCGCAGAAACTGAAATTTCGTGAACCGCAGCGTGAAGAACGTATTTGTCTGGTTACCACCAATTTTCAGACCAAAAGTATGAGCAGTATGGTTAGTGATACCAGTTGTACCTTTCCGAGCAGCGATGGTACATTTTGGAAACATTGGATTCAGACCAAAGATGGCCAGTGCGGTAATCCGCTGGTGAGTACCCGTGATGGCTTTATTGTTGGTATTCATAGCGCCAGCAATTTTACCAATACCAATAATTATTTCGCAAGCGTGCCGAAAAATTTTATGGAACTGCTGACCAATCAGGAAGCACAGCAGTGGGTGAGTGGCTGGCGTCTGAATGCAGATAGCGTGCTGTGGGGTGGTCATAAAGTTTTTATGAATAAGCCGGAAGAACCGTTTCAGCCGGTTAAAGAAGCCACCCAGCTGATGAATGGTAGCGGCAGCTGGAGTCATCCGCAGTTTGAAAAAGGTGGCGGCAGTCATCATCATCATCACCATTAA。 2. An amino acid molecule encoding the TEV protease according to claim 1, characterized in that Its amino acid sequence is shown in SEQ ID NO:2: MRGSGESLFKGPRDYNPISSSICHLTNESDGHTTSLYGIGFGPFIITNKHLFRRNNGTLLVQSLHGVFKVKDTTTLQQHLVDGRDMIIIRMPKDFPPFPQKLKFREPQREERICLVTTNFQTKSMSSMVSD TSCTFPSSDGTFWKHWIQTKDGQCGNPLVSTRDGFIVGIHSASNFTNTNNYFASVPKNFMELLTNQEAQQWVSGWRLNADSVLWGGHKVFMNKPEEPFQPVKEATQLMNGSGSWSHPQFEKGGGSHHHHHH.

3. A method for soluble expression of TEV protease in Escherichia coli, characterized in that: The steps are: Step 1: inserting the TEV protease gene sequence shown in SEQ ID NO: 1 between the restriction endonucleases of the PET-32a vector to obtain the recombinant plasmid pET32a-TEV; Step 2: transformation: add 1 μl of recombinant plasmid pET32a-TEV to BL21 (DE3) competent cells, place on ice for 30 min, then heat shock at 42°C for 60 s; place on ice for 3 min; then add 500 μl of LB in a clean bench, place on a 37°C shaker at 200 rpm / min for 1 h to activate the bacteria; pour the activation solution into a plate with kanamycin resistance, seal the plate after the liquid is air-dried, and place in a 37°C incubator overnight; Step 3: Activation, expansion, and induction: Pick the cultured monoclonal bacteria and add them to LB liquid culture medium containing 100 μg / mL ampicillin for activation overnight; take 5 mL of the activated bacterial solution, inoculate it into 500 mL of LB liquid culture medium containing 100 μg / mL ampicillin at a volume ratio of 1:100, and shake culture at 37°C and 200 rpm / min until the OD600 value = 0.6; when OD600 = 0.6, add 400 μl of 0.5M IPTG, and induce expression at 23°C and 200 rpm / min for 16 hours to obtain the induced bacterial solution; Step 4: Resuspend and break: pour the induced bacterial liquid into the collection bottle, balance, centrifuge at 5000rpm / min for 15min, and obtain 2g of bacterial cells after centrifugation; resuspend with breaking buffer; the collected bacterial cells after resuspending are broken by low-temperature ultrasonic until the bacterial cells are clear. After breaking, centrifuge at 4℃ and 12000rpm / min for 30min, collect the supernatant, and filter the collected supernatant through a 0.45um filter membrane; Step 5, affinity chromatography: add the filtered supernatant to a nickel chromatography column containing Ni-NTA agarose gel, load the sample after equilibration with a crushing buffer for 5CV, wash the impurities with 8CV crushing buffer, then elute the impurities with 6CV 10% eluent, and finally elute the target protein with a 10CV gradient elution to obtain TEV protease; Step 6, SDS-PAGE detection: collect TEV protease and perform SDS-PAGE to detect the purity of the purified protein; Step 7, desalting: adding TEV protease to the desalting column, equilibrate with desalting buffer for 5CV before loading, eluting with 1CV desalting buffer to remove imidazole in TEV protease to obtain desalted TEV protease; Step 8, TEV enzyme activity determination: The concentration of TEV protease after desalting was determined, and the enzyme activity test was performed. W63 containing TEV protease cleavage site and TEV enzyme were mixed at a molar ratio of 1:10, reacted at 4°C overnight, and the enzyme cleavage effect was detected by SDS-PAGE.

4. The method for soluble expression of TEV protease in Escherichia coli according to claim 3, characterized in that: The disruption buffer consisted of 50 mM Tris pH 8.0 and 300 mM NaCl.

5. The method for soluble expression of TEV protease in Escherichia coli according to claim 3, characterized in that: The 10% eluent consisted of 50 mM Tris pH 8.0, 300 mM NaCl and 50 mM imidazole.

6. The method for soluble expression of TEV protease in Escherichia coli according to claim 3, characterized in that: The desalting buffer consisted of 20 mM Tris pH 8.0 and 100 mM NaCl.

7. The method for soluble expression of TEV protease in Escherichia coli according to claim 3, characterized in that: The system buffer for the eighth step reaction consists of 20 mM Tris-HCl (pH 8.0), 100 mM NaCl and 1 mM DTT.

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