A modular set of prokaryotic and eukaryotic expression vectors
A modular set of prokaryotic and eukaryotic expression vectors
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DOI：
10.1006/abio.1999.4383
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发表时间：
2000-01-01
影响因子：
2.9
通讯作者：
Melcher, K
中科院分区：
文献类型：
--
作者：
Melcher, K
为了提高重组融合蛋白的亲和力纯化，描述了一系列模块化的通用表达载体。这些载体的特殊特征包括：(I)即使在非常低的表达率的情况下也能产生极其纯净的蛋白质的系列亲和标签(Hexahistidine-GST)，(Ii)在各种条件下由标记有Hexahistidine的烟草蚀刻病毒(TEV)蛋白酶高效地切割亲和标签，(Iii)通过在蛋白质的N-末端仅用一个(单个甘氨酸)或两个(Gly-Ala)氨基酸进行蛋白质水解性切割的PCR克隆设计，以及(Iv)在大肠杆菌或酿酒酵母中表达，此外，在变性条件下，可以产生单一的六组氨酸标记的蛋白质用于纯化，一些载体允许增加五个氨基酸激酶识别位点，以便于对蛋白质进行放射性标记。为了说明这些载体的用途，我们在天然或变性条件下生产和纯化了酵母半乳糖调节子的所有调控成分，而不是丰富的高度可溶性蛋白，并证实了它们的生物学活性。(C)2000年学术出版社。
A modular series of versatile expression vectors is described for improved affinity purification of recombinant fusion proteins. Special features of these vectors include (i) serial affinity tags (hexahistidine-GST) to yield extremely pure protein even with very low expression rates, (ii) highly efficient proteolytic cleavage of affinity tags under a variety of conditions by hexahistidine-tagged tobacco etch virus (TEV) protease, (iii) PCR cloning design that results in a product of proteolytic cleavage with only one (a single glycine) or two (gly-ala) amino acids at the N-terminus of the protein, and (iv) expression in either Escherichia coli or Saccharomyces cerevisiae, In addition, singly hexahistidine-tagged proteins can be produced for purification under denaturing conditions and some vectors allow addition of five amino acid kinase recognition sites for easy radiolabeling of proteins. To illustrate the use of these vectors, all regulatory components of the yeast GAL regulon, rather than abundant highly soluble proteins, were produced and purified under native or denaturing conditions, and their biological activity was confirmed. (C) 2000 Academic Press.