A novel strategy for stabilization of Escherichia coli ribonuclease HI involving a screen for an intragenic suppressor of carboxyl-terminal deletions

Mitsuru Haruki, Eriko Noguchi, Atsuko Akasako, Motohisa Oobatake, Mitsuhiro Itaya, Shigenori Kanaya

研究成果: Article査読

38 被引用数 (Scopus)

抄録

A strategy to genetically select Escherichia coli ribonuclease HI mutants with enhanced thermostability is described. E. coli strain MIC3001, which shows an RNase H-dependent, temperature-sensitive growth phenotype, was used for this purpose. Introduction of the rnhA gene permits the growth of this temperature-sensitive strain, whereas the gene for the truncated protein, 142-RNase HI, which lacks the carboxyl-terminal 13 residues, cannot. Analyses of the production levels and the stability of a series of mutant proteins with COOH-terminal truncations suggested that 142-RNase HI is nonfunctional in vivo because of a dramatic decrease in the protein stability. Polymerase chain reaction-mediated random mutagenesis of the rnhA142 gene, encoding 142- RNase HI, followed by selection of revertants, allowed us to isolate 11 single amino acid substitutions that render 142-RNase HI functional in vivo. Of them, eight substitutions were shown to enhance the thermal stability of the wild-type RNase HI protein, and of these, six were novel. The genetic selection strategy employed in this experiment was thus shown to be effective for identifying amino acid substitutions that enhance the thermal stability of E. coli RNase HI. Such a strategy would be versatile if a protein of interest could be destabilized by a deletion or a truncation and a conditional-lethal strain were available.

本文言語English
ページ(範囲)26904-26911
ページ数8
ジャーナルJournal of Biological Chemistry
269
43
出版ステータスPublished - 1994 10 28

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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