Expression and purification of a spider silk protein: A new strategy for producing repetitive proteins
Expression and purification of a spider silk protein: A new strategy for producing repetitive proteins
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DOI：
10.1006/prep.1996.0060
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发表时间：
1996-06-01
影响因子：
1.6
通讯作者：
Fournier, MJ
中科院分区：
文献类型：
--
作者：
Lewis, RV;Hinman, M;Fournier, MJ
合成基因是根据蜘蛛拖丝丝蛋白MaSp 2的已知序列构建的。这些基因具有8、16或32个连续单位的蛋白质的共有重复序列。这些人工基因的构建使用的策略涉及兼容的，但不可再生的限制性位点，这使得非常大的插入在一个精确控制的方式建设。这种策略在由相同或不同重复单元组成的重复蛋白质的受控构建中具有普遍实用性。来自16个单元重复的蛋白质在大肠杆菌中以高达10 mg/g细胞湿重的水平产生，尽管1-2 mg/g的产量更典型。使用亲和柱以高回收率容易地纯化蛋白质。纯化的蛋白具有预测的氨基酸组成和切割前导序列后的N-末端序列。所描述的方法将允许生产足够量的蛋白质用于基本结构/功能研究，包括合成纤维的生产。(C)出版社：Academic Press，Inc.
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.