By the aim of developing expression system for Bacillus subtilis (B. subtilis), a series of strong promoters was constructed by the improvements of the -35 element, the -10 element, the -15 region, UP element, transcriptional start site, stabilizing element and Shine-Dalgamo (SD) sequence from Pgrac promoter, which have had high expression levels of recombinant proteins using Isopropyl P-D-I-thiogalactopyranoside (IPTG) as an inducer. Among them Pgrac I 00 which is an outstanding promoter allows expressing BgaB up to 30 percent. This promoter was modified at UP element, -35 consensus region and -15 region in comparison with Pgrac promoter. Pgrac100 system includes pHT253 plasmid (Pgrac100-8xHis-MCS) containing His-tag at Nterminal and pHT254 plasmid (pgrac100-MCS-8xHis) containing His-tag at C-terminal. However, the influence of His-tag on expression of target protein has not been investigated. In this study, the authors use beta-galacosidase (BgaB) as a reporter protein to investigate the influence of His-tag on the expression of BgaB in the fusion form at the Nor C-terminal in B. subtilis. Plasmid pHT1178 (His-BgaB) harboring the reporter gene fused with His-tag at the N terminal was constructed. Three plasmids, pHT100 (BgaB), pHT1178 and pHT1179 (BgaB-His) were transformed into B. subtilis 1012 and the expression levels of BgaB were measured by activity-based assay and SDS-PAGE. The results showed that the His-tag which fused at N-terminal reduced significantly the expression of BgaB in B. subtilis. The study concerning the expression levels of the reporter protein fused at the N or C will provide an option for selection of suitable expression vector for production of recombinant proteins in B. subtilis.
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