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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Design of temperature-sensitive mutants solely from amino acid sequence.
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Design of temperature-sensitive mutants solely from amino acid sequence.

机译：仅从氨基酸序列设计温度敏感性突变体。

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摘要

Temperature-sensitive (Ts) mutants are a powerful tool with which to study gene function in vivo. Ts mutants are typically generated by random mutagenesis followed by laborious screening procedures. By using the Escherichia coli cytotoxin CcdB as a model system, simple procedures for generating Ts mutants at high frequency through site-directed mutagenesis were developed. Putative buried, hydrophobic residues are selected through analysis of the protein sequence. Residue burial is confirmed by ensuring that substitution of the residue by Asp leads to protein inactivation. At such sites, a Ts phenotype can typically be generated either by (i) substitution of two predicted, buried residues with the 18 remaining amino acids or (ii) introduction of Lys, Ser, Ala, and Trp at three to four predicted buried sites. By using these design strategies, 17 tight Ts mutants of CcdB were isolated at four predicted buried sites. The rules were further verified by making several Ts mutants of yeast Gal4 at residues 68,69, and 70. No Ts mutants of either protein have been previously reported. Such Ts mutants of Gal4 can be used for conditional expression of a variety of genes by using the well characterized upstream-activating-sequence-Gal4 system.
机译：温度敏感（Ts）突变体是用于研究体内基因功能的强大工具。 Ts突变体通常是通过随机诱变然后进行费力的筛选程序生成的。通过使用大肠杆菌细胞毒素CcdB作为模型系统，开发了通过定点诱变高频产生Ts突变体的简单程序。通过对蛋白质序列的分析来选择推定的掩埋疏水残基。通过确保残基被Asp取代导致蛋白质失活来确认残骸的埋葬。在这样的位点，通常可以通过（i）用18个剩余氨基酸替换两个预测的，埋藏的残基或（ii）在三到四个预测的埋藏位点引入Lys，Ser，Ala和Trp来生成Ts表型。 。通过使用这些设计策略，在四个预测的掩埋位点分离了17个紧密的CcdB Ts突变体。通过在残基68,69和70处制备酵母Gal4的多个Ts突变体进一步验证了该规则。以前没有报道过这两种蛋白质的Ts突变体。通过使用特征明确的上游激活序列-Gal4系统，Gal4的此类Ts突变体可用于条件表达多种基因。

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