Combinatorial cassette mutagenesis as a probe of the informational content of protein sequences.
Combinatorial cassette mutagenesis as a probe of the informational content of protein sequences.
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DOI：
10.1126/science.3388019
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发表时间：
1988-07
期刊：
影响因子：
56.9
通讯作者：
J. Reidhaar-Olson;Robert T. Sauer
中科院分区：
文献类型：
--
作者：
J. Reidhaar-Olson;Robert T. Sauer
设计了一种组合盒式诱变方法，以容易地确定蛋白质序列中单个残基的信息含量。该技术包括通过寡核苷酸盒诱变同时随机化两个或三个位置，选择功能蛋白，然后测序以确定每个位置上允许的取代谱。这种方法的重复应用于λ阻遏物的DNA结合结构域的二聚体界面揭示了在每个位置允许的取代的数量和类型是极其可变的。在某些位置，只有一个或两个残基是功能上可接受的;在其他位置，容许广泛的残基和残基类型。在每个位置处允许的取代的数目大致与野生型侧链的溶剂可及性相关。
A method of combinatorial cassette mutagenesis was designed to readily determine the informational content of individual residues in protein sequences. The technique consists of simultaneously randomizing two or three positions by oligonucleotide cassette mutagenesis, selecting for functional protein, and then sequencing to determine the spectrum of allowable substitutions at each position. Repeated application of this method to the dimer interface of the DNA-binding domain of lambda repressor reveals that the number and type of substitutions allowed at each position are extremely variable. At some positions only one or two residues are functionally acceptable; at other positions a wide range of residues and residue types are tolerated. The number of substitutions allowed at each position roughly correlates with the solvent accessibility of the wild-type side chain.