大肠杆菌CFT073中Curli系统重要基因csgF的克隆、表达、纯化和结构分析

Expression, purification and structural analysis of csgF gene of curli systems from Escherichia coli CFT073

摘要:

【目的】对大肠杆菌CFT073中Culri系统的重要蛋白CsgF进行高效表达，探索其纯化条件和三维结构，为研究Curli生物合成机制提供理论基础。【方法】以大肠杆菌CFT073基因组为模板扩增csgF基因，构建pET28a-csgF(nsp)-N-6His、pET28a-csgF(20?129)-N-6His、pET28a-csgF-C-6His和pET28a-csgF(nsp)-C-6His等重组质粒，转化到大肠杆菌DH5α并在BL21(DE3)中诱导表达；通过十二烷基磺酸钠-聚丙烯酰胺凝胶电泳鉴定CsgF蛋白在大肠杆菌中的表达情况，用Ni-NTA His Bind Resin和凝胶排阻层析色谱纯化重组蛋白CsgF，SDS-PAGE和Western blotting 方法鉴定分析；用Pull down实验研究CsgF与CsgG蛋白的相互作用，同源模建方法分析重组蛋白CsgF的三级结构。【结果】克隆了目的基因csgF，并筛选出稳定CsgF蛋白的条件：50 mmol/L sodium acetate (pH 5.0)、150 mmol/L NaCl、5% Glyercol；CsgF与CsgG存在相互作用，CsgF三维结构模型显示为(β/α)。【结论】获得了高纯度稳定的CsgF重组蛋白及其三维结构，为进一步研究CsgF结构与功能奠定了基础。

关键词:大肠杆菌CFT073，CsgF，基因克隆，蛋白质表达纯化，凝胶排阻层析色谱

Abstract:

[Objective] In this experiment we explored the expression, purification condition and tertiary structure of the Curli family protein CsgF of Escherichia coli CFT073, to provide a theoretical basis for studying the biosynthetic mechanism of Curli. [Methods] The csgF gene was amplified by PCR from E. coli CFT073 genomic DNA, and the recombinant plasmids such as pET28a-csgF(nsp)-N-6His, pET28a-csgF(20?129)-N-6His, pET28a-csgF-C-6His, and pET28a-csgF(nsp)-C-6His, were constructed. These were then transformed into E. coli strain DH5α, and the expression of the csgF gene in E. coli BL21(DE3) was induced by isopropyl β-D-1-Thiogalactopyranoside (IPTG). Next, CsgF was separated and purified by Ni-chelating affinity chromatograph and gel exclusion chromatography, and was determined by SDS-PAGE and Western blotting assay. The protein interaction of CsgF and CsgG was studied by pull down experiments. The tertiary structure of CsgF was constructed based on homology modeling. [Results] The purified CsgF protein of high stability was obtained in condition of 50 mmol/L sodium acetate (pH 5.0), 150 mmol/L NaCl and 5% glycerol. Pull down experiments showed there was interaction between CsgF and CsgG. Homology modeling demonstrated that the tertiary structure of CsgF was of (β/α) model. [Conclusion] Our findings can lay a foundation for studying the structure and function of CsgF.

Key words:Escherichia coli CFT073, CsgF, Gene cloning, Protein expression and purification, Gel exclusion chromatography