【摘要】 在基因工程的操作中,包涵体的形成是人们利用大肠杆菌表达外源蛋白质的一大难点.本研究将环状芽孢杆菌(Bacillus circulans)环状糊精糖基转移酶(CGTase)的淀粉粒结合域(SBD)基因编码序列的两个拷贝通过一连接肽连接(SBD2)后,克隆到大肠杆菌表达载体pTrcHis B上,得到的pTrcHis B/SBD2质粒转化大肠杆菌Top10,研究了不同培养基、不同诱导温度和时间对SBD2蛋白表达的影响.结果表明,利用相容性溶质山梨醇和甜菜碱等,在高盐胁迫下,实现了SBD2重组蛋白质在大肠杆菌中的可溶性表达,这一结果为在体外研究SBD2蛋白的功能奠定了基础.更多还原

【Abstract】 An artificial tandm repeat of a starch-binding domain(SBD2) was engineered by two copies of the SBD derived from Bacillus circulans cyclodextrin glycosyltransferase via a linker peptide.The SBD2 gene was inserted into an E.coli expression vector,pTrcHis B,to obtain the pTrcHis B/SBD2 vector.Recombinant E.coli Top 10 cells,containing the pTrcHis B/SBD2construct,were grown in LB with 50μg/mL ampicillin and LB with 50μg/mL ampicillin,1mol/L sorbitol and 2.5mmol/L glycyl betaine,respectively.Expression of SBD2 protein was induced by adding of IPTG and analyzed by SDS-PAGE.The results show that large amounts of soluble protein were obtained by growing and inducing the cells under osmotic stress in the presence of sorbitol and glycyl betaine.更多还原