Insertion of foreign random sequences of 120 amino acid residues into an active enzyme.
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.
Doi
N
N
Graduate School of Environmental Earth Sciences, Hokkaido University, Sapporo, Japan.
Itaya
M
M
Yomo
T
T
Tokura
S
S
Yanagawa
H
H
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology*; D015723:Gene Library; D008961:Models, Structural; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D016914:Ribonuclease H / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*
D011994:Recombinant Proteins; D016914:Ribonuclease H; C078856:ribonuclease HI