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

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【作者】 肖谷田谢毅张婕孙筱清吴小舟陈小央王启松

【Author】 XIAO Gutian XIE Yi , ZHANG JieSUN Xiaoqing WU Xiaozhou , CHEN Xiaoyangand WANG Qisong(State Key Laboratory of Genetic Engineering, Institute of Genetics, Fudan University, Shanghai 200433, China)

【机构】 State Key Laboratory of Genetic EngineeringInstitute of GeneticsFudan UniversityShanghai 200433ChinaChina

【摘要】 <正> 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.

【Abstract】 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.

【基金】 Project supported by United Nations Industrial Development Organization (UNIDO).
  • 【文献出处】 Science in China(Series C:Life Sciences) ,中国科学(C辑:生命科学)(英文版) , 编辑部邮箱 ,1997年04期
  • 【分类号】Q343
  • 【下载频次】9
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