铜绿假单胞菌的LexA蛋白在大肠杆菌中的高效表达与纯化

目的:对铜绿假单胞菌(Pseudomonas aeruginosa,PA)的阻遏蛋白LexA进行基因克隆、重组表达及蛋白纯化.方法:采用PCR法从PAO1株基因组中扩增出1 086 bp的LexA基因,将此基因片段插入到表达载体pET32a( )上,并在大肠杆菌BL21(DE3)中表达.包涵体洗涤后用8 mol/L尿素溶解,以镍离子亲合柱层析作为第1步纯化,Superdex75凝胶过滤层析作为第2步精细纯化,反相高相液相色谱法(HPLC)测定蛋白的浓度.结果:LexA以包涵体形式表达,表达量为45%,经镍离子亲合柱层析纯化后LexA蛋白纯度达到85%以上,回收率大于80%,镍离子亲合柱层析和凝胶过滤层析两步纯化目的蛋白,经HPLC测定,最终目的蛋白的纯度为98.97%.结论:在pET32a( )表达系统中实现了PA的LexA蛋白的高效表达,两步纯化法获得高纯度的目的蛋白,为进一步鉴定LexA靶位点奠定了基础.

High level expression and purification of LexA from Pseudomonas aeruginosa in E. coli

Aim:To clone LexA gene,expression and purify the repressor LexA from Pseudomonas aeruginosa(PA). Methods:The genomic DNA was extracted from the PAO1,the gene fragment was encoding the mature LexA was amplied by PCR.It was linked the vector pET32a(+) and expressed in the E.coli BL21(DE3).The expressed protein was purified by two steps of Ni2+ chelate affinity chromatography and gel filtration chromatography sequentially.Results:The expressed fused protein was found in insoluble form,accounted 45% of the total protein.The purity was 80%,the recovery rate was over 80%.The final purity was 98.97%,which was determined by the HPLC.Conclusion:LexA of PA is highly expressed in E.coli,and purified interest protein is obtained,which provide the foundation for the further identification on target site of LexA.