Stephens Linda L, Shonhai Addmore, Blatch Gregory L
Department of Biochemistry, Microbiology and Biotechnology, Rhodes University, South Africa.
Protein Expr Purif. 2011 Jun;77(2):159-65. doi: 10.1016/j.pep.2011.01.005. Epub 2011 Jan 22.
Molecular chaperones have been used for the improved expression of target proteins within heterologous systems; however, the chaperone and target protein have seldom been matched in terms of origin. We have developed a heterologous co-expression system that allows independent expression of the plasmodial chaperone, PfHsp70, and a plasmodial target protein. In this study, the target was Plasmodium falciparum GTP cyclohydrolase I (PfGCHI), the first enzyme in the plasmodial folate pathway. The sequential expression of the molecular chaperone followed by the target protein increased the expression of soluble functional PfGCHI. His-tagged PfGCHI was successfully purified using nickel affinity chromatography, and the specific activity was determined by high performance liquid chromatography with spectrofluorometeric detection to be 5.93nmol/h/mg. This is the first report of a heterologous co-expression system in which a plasmodial chaperone is harnessed for the improved production and purification of a plasmodial target protein.
分子伴侣已被用于在异源系统中提高靶蛋白的表达；然而，伴侣蛋白和靶蛋白在来源方面很少匹配。我们开发了一种异源共表达系统，该系统允许独立表达疟原虫伴侣蛋白PfHsp70和疟原虫靶蛋白。在本研究中，靶标是恶性疟原虫GTP环水解酶I（PfGCHI），它是疟原虫叶酸途径中的第一种酶。分子伴侣随后靶蛋白的顺序表达增加了可溶性功能性PfGCHI的表达。His标签的PfGCHI通过镍亲和层析成功纯化，其比活性通过高效液相色谱-荧光检测法测定为5.93nmol/h/mg。这是首次报道利用疟原虫伴侣蛋白改进疟原虫靶蛋白的生产和纯化的异源共表达系统。
