Expression and purification of a spider silk protein: A new strategy for producing repetitive proteins

被引:134
作者
Lewis, RV [1 ]
Hinman, M [1 ]
Kothakota, S [1 ]
Fournier, MJ [1 ]
机构
[1] UNIV MASSACHUSETTS,DEPT BIOCHEM & MOLEC BIOL,AMHERST,MA 01003
基金
美国国家科学基金会;
关键词
D O I
10.1006/prep.1996.0060
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
Synthetic genes were constructed based on the known sequence of the spider dragline silk protein MaSp 2. The genes had 8, 16, or 32 contiguous units of the consensus repeat sequence of the protein. These artificial genes were constructed using a strategy involving compatible but nonregenerable restriction sites, which allowed construction of very large inserts in a precisely controlled manner. This strategy should have general utility in the controlled construction of repetitive proteins composed of identical or different repeat units. The protein from the 16-unit repeat was produced in Escherichia coli at levels up to 10 mg/g wet wt of cells although yields of 1-2 mg/g were more typical. The protein was easily purified with high recovery using an affinity column. The purified protein had the predicted amino acid composition and N-terminal sequence after cleavage of a leader sequence. The methodology described will allow production of sufficient quantities of protein for basic structure/function studies including production of synthetic fibers. (C) 1996 Academic Press, Inc.
引用
收藏
页码:400 / 406
页数:7
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