Nghiên cứu nâng cao hiệu suất biểu hiện tev protease trong vi khuẩn escherichia coli bằng protein dung hợp super folder green fluorescent protein (sfgfp)

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Tác giả: Văn Quyền Đồng, Thị Hiền Vũ

Ngôn ngữ: vie

Ký hiệu phân loại: 570.78 Life sciences Biology

Thông tin xuất bản: Công nghệ Sinh học, 2014

Mô tả vật lý: 143-149

Bộ sưu tập: Metadata

ID: 564980

The use of fusion tags has become a widely practice in the production of recombinant proteins. However, in many cases tags can interfere with the functional or biological activity of its fusion partner. For this reason, it is genrally advisable to remove tags from recombinant proteins. TEV protease (TEVp), the 27 kDa catalytic domain of the nuclear inclusion a (NIa) protease from tobacco etch virus, was widely used .to remove fusion tags from recombinant proteins because of its stringent sequence specificity. However, expression of TEV protease in E. coli has been problematic due to its low solubility. To overcome this obstacle, many strategies have been addressed such as designing a more stable mutant of TEVp, co-expression with chaperone, lowtemperature expression and fusing to other proteins. In this study, the authors introduced a method to enhance expression of TEVp by using visible superfolder green fluorescent protein (sfGFP) as the fPsion tag. sfGFP, a variant ofGFP, has proven to be able to rapidly fold and mature even when fused to poorly folding peptides, therefore improving the solubility, stability and expression level of the fusion protein. Gene encoding for sfGFP-TEVp was made by overlap extension PCR. cloned into the expression vector pET21-a(+) and then transformed into E. coli strain BL21 (DE3) to overexpress the target protein. The results indicate that, recombinant sfGFP-TEVp optimally expressed at temperatures of 25°C, ImM IPTG and after 12 hours of induction. The recombinant TEVp was successfully purified by Ni-NTA affinity chromatography column and yield of over 8 mg per 100 ml culture which was 8 fold increased compared to that of the original TEV protease. Significantly, sfGFP- TEVp is expressed in soluble form and has specific catalytic activity toward its substrate.
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