A thermoresistant mutant of ribonuclease t1 having three disulfide bonds

Satoshi Nishikawa, Jeanne Adiwinata, Hiroshi Morioka, Takao Fujimura, Toshiki Tanaka, Seiichi Uesugi, Toshio Hakoshima, Ken Ichi Tomita, Setsuko Nakagawa, Morio Ikehara

Research output: Contribution to journalArticlepeer-review

14 Citations (Scopus)

Abstract

Molecular-dynamic calculations predict that, if Tyr24 and Asn84 are each replaced by a Cys residue, it should be possible to form a third disulfide bond in ribonuclease T1 (RNase T1) between these residues, with only minimal conformational changes at the catalytic site. The gene encoding such a mutant variant of RNase T1 (Tyr24 - Cys24, Asn84 - Cys84) was constructed by the cassette mutagenesis method using a chemically synthesized gene. In order to reduce the toxic effect of the mutant enzyme (RNase T1S) on an Escherichia coli host, we arranged for the protein to be secreted into the periplasmic space by using a vector that harbors a gene for an alkaline phosphatase signal peptide under the control of the trp promoter. The nucleolytic activity of RNase T1S toward pGpC was approximately the same as that of RNase T1 at 37°C (pH 7.5). Moreover, at 55°C, RNase T1S retained nearly 70% of its activity while the activity of the wild-type enzyme was reduced to <10%. RNase T1S was also more resistant to denaturation by urea than the wild-type enzyme. However, unlike RNase T1, RNase T1S was irreversibly and almost totally inactivated by boiling at 100°C for 15 min.

Original languageEnglish
Pages (from-to)443-448
Number of pages6
JournalProtein Engineering, Design and Selection
Volume3
Issue number5
DOIs
Publication statusPublished - 1 Apr 1990

Keywords

  • Disulfide bond
  • Irreversible inactivation
  • Ribonuclease Tl
  • Thermostability

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