Insertion of foreign random sequences of 120 amino acid residues into an active enzyme
Insertion of foreign random sequences of 120 amino acid residues into an active enzyme
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DOI：
10.1016/s0014-5793(96)01522-0
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发表时间：
1997-02-03
期刊：
影响因子：
3.5
通讯作者：
Yanagawa, H
中科院分区：
文献类型：
--
作者：
Doi, N;Itaya, M;Yanagawa, H
将120-130个氨基酸残基的随机序列插入大肠杆菌RNase HI的表面环区。对该文库进行筛选，发现约10%的克隆保留RNase H活性。随后的随机诱变导致RNase H活性和蛋白质溶解度的增加。插入的区域仅仅被发现对突变蛋白的二级结构没有贡献。柔性序列的高频率插入和通过进一步诱变增加蛋白质的功能模拟了蛋白质进化中的事件之一。
Random sequences of 120-130 amino acid residues mere inserted into a surface loop region of Escherichia coli RNase HI. This library mas screened and about 10% of the clones were found to retain RNase H activity. Subsequent random mutagenesis led to an increase in RNase H activity and solubility of the protein. The inserted regions mere found not to contribute to the secondary structure of the mutant protein. The high frequency of insertion of flexible sequences and the increase in the protein's function by further mutagenesis simulate one of the events in protein evolution.