使用内含肽-几丁质结合结构域（CBD）系统克隆、表达和纯化模仿葡萄球菌溶葡萄球菌素
Cloning, expression, and purification of the Staphylococcus simulans lysostaphin using the intein-chitin-binding domain (CBD) system.
作者信息
Szweda P, Pladzyk R, Kotlowski R, Kur J
机构信息
Department of Microbiology, Technical University of Gdańsk, ul. Narutowicza 11/12, 80-952 Gdańsk, Poland.
出版信息
Protein Expr Purif. 2001 Aug;22(3):467-71. doi: 10.1006/prep.2001.1454.
The Staphylococcus simulans gene encoding lysostaphin has been PCR amplified from pRG5 recombinant plasmid (ATCC 67076) and cloned into Escherichia coli expression pTYB12 vector (IMPACT-CN System, New England BioLabs) which allows the overexpression of a target protein as a fusion to a self-cleavable affinity tag. The self-cleavage activity of the intein allows the release of the lysostaphin enzyme from the chitin-bound intein tag, resulting in a single-column purification of the target protein. This abundant overproduction allows purifying milligram amounts of the enzyme.
编码溶葡萄球菌酶的模仿葡萄球菌基因已通过聚合酶链反应（PCR）从pRG5重组质粒（ATCC 67076）中扩增出来，并克隆到大肠杆菌表达载体pTYB12（IMPACT-CN系统，新英格兰生物实验室）中，该载体可使目标蛋白作为与可自我切割的亲和标签融合的蛋白进行过量表达。内含肽的自我切割活性可使溶葡萄球菌酶从与几丁质结合的内含肽标签上释放出来，从而实现目标蛋白的单柱纯化。这种大量的过量生产使得能够纯化出毫克量的该酶。
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