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 Biotechnoljournal_title
Nature biotechnologyauthors
Matsuura T,Miyai K,Trakulnaleamsai S,Yomo T,Shima Y,Miki S,Yamamoto K,Urabe Idoi
10.1038/5232subject
Has Abstractpub_date
1999-01-01 00:00:00pages
58-61issue
1eissn
1087-0156issn
1546-1696journal_volume
17pub_type
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