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Insertion of foreign random sequences of 120 amino acid residues into an active enzyme
Author(s) -
Doi Nobuhide,
Itaya Mitsuhiro,
Yomo Tetsuya,
Tokura Seiichi,
Yanagawa Hiroshi
Publication year - 1997
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(96)01522-0
Subject(s) - mutagenesis , rnase p , amino acid , mutant , site directed mutagenesis , escherichia coli , s tag , biochemistry , directed evolution , chemistry , enzyme , protein engineering , biology , gene , rna
Random sequences of 120–130 amino acid residues were inserted into a surface loop region of Escherichia coli RNase HI. This library was screened and about 10% of the clones were found to retain RNase H activity. Subsequent random mutagenesis led to an increase in RNase H activity and solubility of the protein. The inserted regions were found not to contribute to the secondary structure of the mutant protein. The high frequency of insertion of flexible sequences and the increase in the protein's function by further mutagenesis simulate one of the events in protein evolution.