A semi-rational design strategy of directed evolution combined with chemical synthesis of DNA sequences.

A semi-rational design strategy of directed evolution combined with chemical synthesis of DNA sequences. Research Abstract Details 

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A semi-rational design strategy of directed evolution combined with chemical synthesis of DNA sequences. Abstract Text:

Ai-Sheng Xiong,Ri-He Peng,Jing Zhuang,Jin-Ge Liu,Feng Gao,Fang Xu,Bin Cai,Quan-Hong Yao,Ai-Sheng Xiong,Ri-He Peng,Jing Zhuang,Jin-Ge Liu,Feng Gao,Fang Xu,Bin Cai,Quan-Hong Yao,Ai-Sheng Xiong,Ri-He Peng,Jing Zhuang,Jin-Ge Liu,Feng Gao,Fang Xu,Bin Cai,Quan-Hong Yao,

Abstract Directed evolution in vitro is a powerful molecular tool for the creation of new biological phenotypes. It is unclear whether it is more efficient to mutate an enzyme randomly or to mutate just the active sites or key sites. In this study, the strategy of a semi-rational design of directed evolution combined with whole sequence and sites was developed. The 1553 bp gene encoding the thermostable beta-galactosidase of Pyrococcus woesei was chemically synthesized and optimized for G+C content and mRNA secondary structures. The synthesized gene product was used as a template or as a wild-type control. On the basis of the first round of DNA shuffling, library construction and screening, one mutant of YH6754 was isolated with higher activity. Eight potential key sites were deduced from the sequence of the shuffled gene, and 16 degenerate oligonucleotides were designed according to those eight amino acids. Two variants of YG6765 and YG8252 were screened in the second part of DNA shuffling, library construction and screening. For comparison, one mutant of YH8757 was screened through the same routine rounds of directed evolution with YH6754 as template. The purified beta-galactosidase from YH8757 exhibited a lower specific activity at 25 degrees C than those purified from mutated YG6755 and YG8252.

A semi-rational design strategy of directed evolution combined with chemical synthesis of DNA sequences. Publishing Authors By Initials

AS Xiong,RH Peng,J Zhuang,JG Liu,F Gao,F Xu,B Cai,QH Yao,AS Xiong,RH Peng,J Zhuang,JG Liu,F Gao,F Xu,B Cai,QH Yao,AS Xiong,RH Peng,J Zhuang,JG Liu,F Gao,F Xu,B Cai,QH Yao,

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A semi-rational design strategy of directed evolution combined with chemical synthesis of DNA sequences. Journal Published:

PUBLICATION TYPE: Journal Article

Journal: Biological chemistry

VOLUME: 388

Page Numbers: 1291-300

Journal Abbreviation: Biol. Chem.

ISSN: 1431-6730

DAY: 20

MONTH: Dec

YEAR: 2007

A semi-rational design strategy of directed evolution combined with chemical synthesis of DNA sequences. Information

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LANGUAGE: eng

NlmUniqueID: 9700112

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AFFILIATION: 1Biotechnology Research Institute of Shanghai Academy of Agricultural Sciences, 2901 Beidi Road, Shanghai 201106, China.

Country: Germany

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MEDLINETA: Biol Chem

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