细菌脂肪酶在大肠杆菌细胞表面的功能性展示

Display of Functionally Active Lipases on the Escherichia coli Cell Surface

细胞表面展示技术已广泛应用于突变文库的高通量筛选,有力地促进了蛋白质工程的发展。以来自于铜绿假单胞菌的自转运蛋白Est A的羧基端结构域作为锚定区,构建脂肪酶LipA与EstA羧基端结构域的融合基因,并将融合基因插入到改造后的pACYC-Duet表达载体中,获得表面展示载体pBCCB-X1。将载体pBCCB-X1分别导入到大肠杆菌JK321和大肠杆菌UT5600菌株中,以IPTG诱导融合基因的表达。分别用三丁酸甘油酯定性检测和pNPO定量检测诱导表达后的全细胞脂肪酶的水解活性。试验结果表明,脂肪酶LipA在大肠杆菌JK321和大肠杆菌UT5600细胞表面均得到功能性展示,水解活性分别为(2.8±0.1)U/OD和(2.6±0.06)U/OD。脂肪酶LipA在大肠杆菌细胞表面的功能性展示,为后续高通量筛选LipA突变基因文库,奠定了坚实的基础。

Cell-surface display technology was used widely in the filed of high throughput screening of a mutant library, which promoted the development of protein engineering. The carboxyl terminal domain of the EstA from Pseudomonas aeruginosa was used as carrier protein and the lipA gene was fused to the estA’gene by overlap extension PCR. The fusion gene lipA-estA’was then inserted the genetically modified plasmid pACYC-Duet, which promoter was changed into lacZ promoter. The resulting plasmid pBCMB-X1 was transformed into E. coli JK321 and E. coli UT5600, respectively. The lipA gene was induced expression by IPTG and the recombinant LipA was functionally displayed on the cell surface of E. coli JK321 and E. coli.UT5600, respectively. The hydrolysis activity of the LipA was（2.8±0.1）U/OD and（2.6±0.06）U/OD, respectively.