Zhao, Huimin and Giver, Lori and Shao, Zhixin and Affholter, Joseph A. and Arnold, Frances H. (1998) Molecular evolution by staggered extension process (StEP) in vitro recombination. Nature Biotechnology, 16 (3). pp. 258-261. ISSN 1087-0156. doi:10.1038/nbt0398-258. https://resolver.caltech.edu/CaltechAUTHORS:20150618-135325510
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Abstract
We have developed a simple and efficient method for in vitro mutagenesis and recombination of polynu-cleotide sequences. The staggered extension process (StEP) consists of priming the template sequence(s) followed by repeated cycles of denaturation and extremely abbreviated annealing/polymerase-catalyzed extension. In each cycle the growing fragments anneal to different templates based on sequence complementarity and extend further. This is repeated until full-length sequences form. Due to template switching, most of the polynucleotides contain sequence information from different parental sequences. The method is demonstrated by the recombination of two genes encoding thermostable subtilisins carrying two phenotypic markers separated by 113 base pairs and eight other point mutation markers. To demonstrate its utility for directed evolution, we have used StEP to recombine a set of five thermostabilized subtilisin E variants identified during a single round of error-prone PCR mutagenesis and screening. Screening the StEP-recombined library yielded an enzyme whose half-life at 65°C is 50 times that of wild-type subtilisin E.
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| Additional Information: | © 1998 Nature Publishing Group. Received 5 November 1997; accepted 23 January 1998. This work was supported by the US Department of Energy's program in Biological and Chemical Technologies Research within the Office of Industrial Technologies, Energy Efficiency and Renewables. The authors are grateful to Xentaro Miyazaki for helpful discussions and Yongkai Ow for her technical assistance. | |||||||||
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| Subject Keywords: | directed evolution, random mutagenesis, subtilisin | |||||||||
| Issue or Number: | 3 | |||||||||
| DOI: | 10.1038/nbt0398-258 | |||||||||
| Record Number: | CaltechAUTHORS:20150618-135325510 | |||||||||
| Persistent URL: | https://resolver.caltech.edu/CaltechAUTHORS:20150618-135325510 | |||||||||
| Usage Policy: | No commercial reproduction, distribution, display or performance rights in this work are provided. | |||||||||
| ID Code: | 58349 | |||||||||
| Collection: | CaltechAUTHORS | |||||||||
| Deposited By: | Ruth Sustaita | |||||||||
| Deposited On: | 18 Jun 2015 21:38 | |||||||||
| Last Modified: | 10 Nov 2021 22:03 |
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