A kind of mature htra2 mutant n196c and its preparation method and application
A mutant and mature technology, applied in the field of protein engineering and genetics, can solve the problems of expensive, low expression and purification efficiency, and limited protein application
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Embodiment 1
[0027] Embodiment 1: Mature HtrA2 mutant expression vector construction
[0028] This embodiment utilizes overlap extension PCR technology to obtain mature HtrA2 mutants through site-directed mutagenesis biotechnology, specifically including the following operations.
[0029] 1.1. Construction of wild-type mature HtrA2 protein expression vector
[0030] According to the pET21b plasmid (conventional Escherichia coli expression plasmid), the upstream primer F1 (shown in SEQ ID NO.5) and the downstream primer R1 (shown in SEQ ID NO.6) were designed to human HtrA2 eukaryotic expression vector pCMV3-HtrA2-C- His (SinoBiological Inc.) was used as a template for PCR amplification. The reaction conditions were pre-denaturation at 95°C for 5 min, 60°C for 45 s, 72°C for 46 s, and 38 cycles; full extension at 72°C for 8 min, and storage at 4°C; the mature HtrA2 gene (expressed Amino acids 134-458 of the complete HtrA2 protein, the amino acid sequence of the expressed mature HtrA2 prote...
Embodiment 2
[0036] Example 2: Expression of wild-type mature HtrA2 protein and its mutants N196C and D202C
[0037] Inoculate engineering bacteria expressing wild-type mature HtrA2 protein and its mutants N196C and D202C (Escherichia coli after 1.1 to 1.3 in Example 1 were respectively transferred to recombinant plasmids) into LB liquid medium containing 200 μg / ml Amp antibiotics , 37°C, 220rpm cultured overnight to obtain bacterial liquid. Inoculate the bacterial solution into 200ml LB liquid medium containing Amp antibiotics at a ratio of 1:100, shake the bacteria to OD at 37°C and 220rpm 600 = 0.6-0.8, add inducer 1mM IPTG, induce expression at 16°C, 220rpm for 12h; collect the bacterial solution, centrifuge at 4°C, 12000rpm for 10min, wash with pre-cooled PBS three times; dissolve the bacteria in PBS buffer, and use a cell disruptor ( JNBIO) broken resuspension liquid 3-5 times (1200Bar), 4 ℃, 12000rpm centrifuge 10min, collect the supernatant, take a small amount for protein quantif...
Embodiment 3
[0038] Example 3: Purification of wild-type mature HtrA2 protein and its mutant N196C
[0039] This embodiment purifies protein by Ni-NTA (IBA) affinity chromatography, specifically including the following operations.
[0040] The supernatant obtained in Example 2 (expression supernatant of wild-type mature HtrA2 protein and expression supernatant of mature HtrA2 mutant N196C) was collected, and after fully mixing with an equal volume of equilibration buffer (1×PBS buffer plus 10 mM imidazole), 0.22 Filter with a μm sterile filter; after loading the sample at 1ml / min, equilibrate the gel column with equilibration buffer; elute with gradient concentration of imidazole in the equilibration buffer, collect protein samples corresponding to each concentration, identify by SDS-PAGE, and the results Such as figure 2 As shown, A panel is the purification result of mature HtrA2 mutant N196C protein, B panel is the purification result of wild-type mature HtrA2 protein, M in A panel an...
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