TEV protease mutant and coding gene and application thereof
A protease and mutant technology, applied in the field of genetic engineering, can solve the problems of low expression yield and low solubility of TEV protease
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Embodiment 1
[0021] Example 1 Obtaining TEV protease mutants by site-directed mutagenesis
[0022] Primers for site-directed mutagenesis were designed, and the vector containing the protease gene of tobacco plaque virus (pET-TEV) was used as a template for PCR amplification. The reaction conditions were: 94°C for 30s, 55°C for 30s, 72°C for 6.5min, and cycled 30 times. After the PCR product was digested with DpnI, it was electrophoresed on a 1% agarose gel. Gel-cutting and purification, the purified PCR product was subjected to end smoothing and 5′ phosphorylation (P) treatment, and then self-ligation was performed with Ligation Solution I, a highly efficient ligation reagent, transformed into Escherichia coli DH5a competent cells, and the mutant plasmid DNA was extracted. At the same time, the five mutants were named as TEV Q , the triple mutant named TEV F .
Embodiment 2
[0023] Example 2 Mutant Gene Sequencing and Sequence Alignment
[0024] The mutant clone DH5a obtained by the above method was sent to Shanghai Sangon Bioengineering Technology Service Co., Ltd. for sequencing, and the sequencing results were input into BioEdit software to compare the differences in the gene base sequence and protein amino acid sequence before and after the mutation.
[0025] The results showed that there were five mutations in the mutant protein and the original TEV protease. The DNA base and amino acid sequence changes of the mutant are as follows:
[0026] The base changes are: the 50th position is replaced by G (guanine) for C (cytosine), the 166th position is replaced by G (guanine) for T (thymine), and the 202nd position is replaced by G (guanine) for A (adenosine) Purine), the 229th position is replaced by G (guanine) for A (adenine), the 403rd position is replaced by G (guanine) for T (thymine), and the 404th position is replaced by G (guanine) for C ...
Embodiment 3
[0028] Example 3 TEV Q Protease and TEV F Protease Solubility and Expression Comparison
[0029] pET-TEV Q and pET-TEV F The recombinant plasmid was transformed into competent Escherichia coli Rosetta (DE3).
[0030] 1. A small amount of induced expression, compared with TEV Q Protease mutants and TEV F Changes in Protease Solubility:
[0031] (1) Pick a single colony and inoculate it in 5 mL of LB medium (containing 25 mg / mL of kanamycin sulfate and 25 mg / mL of chloramphenicol), and culture overnight at 220 r / min in a constant temperature shaker at 37°C;
[0032] (2) Add IPTG (final concentration: 1.0 mmol / L), and induce expression for 20 h on a constant temperature shaker at 20° C. at 220 r / min.
[0033] (3) Collect the cells with a 1.5 mL doffer tube, wash and resuspend the cells in 0.8 mL lysis buffer.
[0034] (4) The resuspension was ultrasonically crushed and centrifuged, and 120 μL of the supernatant was added to 30 μL of 5×SDS-PAGE loading buffer to prepare a pr...
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