Characterization of random‐sequence proteins displayed on the surface of Escherichia coli RNase HI

FEBS Letters

Doi, Nobuhide¹  Yomo, Tetsuya²  Itaya, Mitsuhiro¹  Yanagawa, Hiroshi¹ 

[1] Mitsubishi Kasei Institute of Life Sciences, Machida, Tokyo 194, Japan;Department of Biotechnology, Osaka University, Suita, Osaka 565, Japan

关键词: Directed evolution;    Evolutionary engineering;    Insertional mutagenesis;    Protein folding;    Protein scaffold;    ANS;    1-anilinonaphthalene-8-sulfonic acid;    CD;    circular dichroism;    ORF;    open reading frame;    PCR;    polymerase chain reaction;   

DOI  :  10.1016/S0014-5793(98)00392-5

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

In a previous study, random-sequence proteins of 120–130 amino acid residues were inserted into the surface loop region of the enzyme, Escherichia coli RNase HI [Doi et al. (1997) FEBS Lett. 402, 177–180]. Here we established that the RNase H activity of the insertion mutants is correlated with their secondary structure contents evaluated by circular dichroism measurement at 222 nm. The random-sequence insert of a mutant enzyme possessing relatively high RNase H activity was detached from the RNase HI scaffold, and its characterization indicated that the random-sequence protein maintains its secondary structure after separation from the scaffold. Thus, the structural features of random-sequence proteins were suggested to be monitored by measuring the activity of the scaffold enzyme into which these proteins have been inserted.

【 授权许可】

Unknown