基于谷氨酸棒杆菌NCgl1221蛋白的新型细菌表面展示系统

【目的】开发一种新型的大肠杆菌表面展示系统,为C末端截短NCgl1221蛋白作为锚定蛋白提供科学依据,丰富并优化细菌表面展示系统。【方法】扩增C末端截短NCgl1221序列和β-淀粉酶基因,构建融合蛋白表达载体。将重组载体PET-NA和空载体PET-28a分别转入Rosetta(DE3)pLysS中,IPTG诱导表达,SDS-PAGE和Western blot鉴定融合蛋白表达情况。将诱导表达菌株进行免疫荧光染色,荧光显微镜观察和流式细胞分析检测β-淀粉酶的展示。酶活测定和淀粉水解分析验证被展示β-淀粉酶的活性。【结果】融合蛋白成功地在大肠杆菌中表达,有活性的β-淀粉酶通过与锚定蛋白C末端的融合被展示在了宿主菌表面,展示β-淀粉酶的重组菌可以水解利用培养基中的淀粉。【结论】成功开发了一种以C末端截短NCgl1221为锚定蛋白的新型大肠杆菌表面展示系统,并以此系统展示了分子量大小为56 kDa的活性酶,为该系统在全细胞催化剂或吸附剂等方面的应用奠定了基础。

A novel bacterial cell-surface display system based on NCgl1221 from Corynebacterium glutamicum

Objective]To develop a novel Escherichia coli cell surface display system by using C-terminally truncated NCgl1221 as the anchoring protein,which greatly enriched or optimized the bacterial displayed systems.Methods]We amplified the sequence of C-terminally truncated NCgl1221 and β-amylase,and constructed the fusion expression vector.Then we transformed the recombinant plasmids PET-NA and PET-28a into Rosetta(DE3)pLysS.The fusion protein expression was induced by IPTG and identified by SDS-PAGE and Western blot analysis.The IPTG induced strains were immunostained and investigated by fluorescence microscope and flow cytometry to detect the displayed β-amylase.Finally,we analyzed the activity of β-amylase and starch hydrolization in order to determine whether the displayed β-amylase has the activity or not.Results]The fusion protein was successfully expressed in E.coli,and the active β-amylase was displayed on the cell surface by fusing it to the C terminus of the anchor.The recombinant strain displaying β-amylase can utilize soluble starch in the medium.Conclusion]A novel E.coli surface display system by using C-terminally truncated NCgl1221 as the anchor motif was successfully developed.The active enzyme with a molecular size of 56 kDa was displayed on E.coli by this system,which provided the basis for the application of the system in whole-cell biocatalyst or biosorbent.