Construction and screening of a multi-point site- specific mutant library of subtilisin E with a set of oligonucleotides  

作　　者：肖谷田 谢毅 张婕 孙筱清 吴小舟 陈小央 王启松 

机构地区：[1]State Key Laboratory of Genetic Engineering, Institute of Genetics, Fudan University, Shanghai 200433, China

出　　处：《Science China(Life Sciences)》1997年第4期337-344,共8页中国科学（生命科学英文版）

基　　金：Project supported by United Nations Industrial Development Organization (UNIDO).

摘　　要：A mutant library of subtilisin E containing random combinations of various mutagenized sites wasconstructed by one-round mutagenesis with 15 mutagenic oligonucleotides. Mutants were screened through dot blot hybridization and DNA sequencing. A single-point mutant (Met 222Ala) and a three-point (Asn 76Asp/Asnl09Ser/ I le 205/Cys) mutant gene from the library were expressed. The mutant proteins exhibited conspicuously improved resistance to oxidation and heat treatment, as reported before. The results show that the library is reliable and very useful for protease subtilisin E engineering.A mutant library of subtilisin E containing random combinations of various mutagenized sites wasconstructed by one-round mutagenesis with 15 mutagenic oligonucleotides. Mutants were screened through dot blot hybridization and DNA sequencing. A single-point mutant (Met 222Ala) and a three-point (Asn 76Asp/Asnl09Ser/ I le 205/Cys) mutant gene from the library were expressed. The mutant proteins exhibited conspicuously improved resistance to oxidation and heat treatment, as reported before. The results show that the library is reliable and very useful for protease subtilisin E engineering.

关 键 词：SITE-DIRECTED MUTAGENESIS multi-point SITE-SPECIFIC MUTAGENESIS protein engineering SUBTILISIN E. 

分 类 号：Q343[生物学—遗传学]