Evolutionary molecular engineering by random elongation mutagenesis.

Abstract:

:We describe a new method of random mutagenesis that employs the addition of peptide tails with random sequences to the C-terminal of enzyme molecules. A mutant population of catalase I from Bacillus stearothermophilus prepared by this method has a diversity in thermostability and enzyme activity equal to that obtained after random point mutagenesis. When a triple mutant of catalase I (I108T/D130N/1222T)-the thermostability of which is much lower than that of the wild type-was subjected to random elongation mutagenesis, we generated a mutant population containing only mutants with higher thermostability than the triple mutant. Some had an even higher stability than the wild-type enzyme, whose thermostability is considered to be optimized. These results indicate that peptide addition expands the protein sequence space resulting in a new fitness landscape. The enzyme can then move along the routes of the new landscape until it reaches a new optimum. The combination of random elongation mutagenesis with random point mutagenesis should be a useful approach to the in vitro evolution of proteins with new properties.

journal_name

Nat Biotechnol

journal_title

Nature biotechnology

authors

Matsuura T,Miyai K,Trakulnaleamsai S,Yomo T,Shima Y,Miki S,Yamamoto K,Urabe I

doi

10.1038/5232

subject

Has Abstract

pub_date

1999-01-01 00:00:00

pages

58-61

issue

1

eissn

1087-0156

issn

1546-1696

journal_volume

17

pub_type

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