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

G Xiao; Y Xie; J Zhang; X Sun; X Wu; X Chen; Q Wang

doi:10.1007/BF02881725

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

Sci China C Life Sci. 1997 Aug;40(4):337-44. doi: 10.1007/BF02881725.

Authors

G Xiao¹, Y Xie, J Zhang, X Sun, X Wu, X Chen, Q Wang

Affiliation

¹ Institute of Genetics, Fudan University, Shanghai, China.

PMID: 18762871
DOI: 10.1007/BF02881725

Abstract

A mutant library of subtilisin E containing random combinations of various mutagenized sites was constructed 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/Asn109Ser/ 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.