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Strategies for the in vitro evolution of protein function: enzyme evolution by random recombination of improved sequences

Moore, Jeffrey C. and Jin, Hua-Ming and Kuchner, Olga and Arnold, Frances H. (1997) Strategies for the in vitro evolution of protein function: enzyme evolution by random recombination of improved sequences. Journal of Molecular Biology, 272 (3). pp. 336-347. ISSN 0022-2836. doi:10.1006/jmbi.1997.1252.

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Sets of genes improved by directed evolution can be recombined in vitro to produce further improvements in protein function. Recombination is particularly useful when improved sequences are available; costs of generating such sequences, however, must be weighed against the costs of further evolution by sequential random mutagenesis. Four genes encoding para-nitrobenzyl (pNB) esterase variants exhibiting enhanced activity were recombined in two cycles of high-fidelity DNA shuffling and screening. Genes encoding enzymes exhibiting further improvements in activity were analyzed in order to elucidate evolutionary processes at the DNA level and begin to provide an experimental basis for choosing in vitro evolution strategies and setting key parameters for recombination. DNA sequencing of improved variants from the two rounds of DNA shuffling confirmed important features of the recombination process: rapid fixation and accumulation of beneficial mutations from multiple parent sequences as well as removal of silent and deleterious mutations. The five to sixfold further enhancement of total activity towards the para-nitrophenyl (pNP) ester of loracarbef was obtained through recombination of mutations from several parent sequences as well as new point mutations. Computer simulations of recombination and screening illustrate the trade-offs between recombining fewer parent sequences (in order to reduce screening requirements) and lowering the potential for further evolution. Search strategies which may substantially reduce screening requirements in certain situations are described.

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Arnold, Frances H.0000-0002-4027-364X
Additional Information:© 1997 Academic Press. Received 10 March 1997, Revised 18 June 1997, Accepted 9 July 1997. The authors thank Dr W. P. C. Stemmer (Maxygen) for many helpful discussions, Dr Steve Queener (Eli Lilly & Co.) for providing us the wild-type pNB esterase and the challenge to evolve it, and Ms Rebecca Little (Eli Lilly & Co.) for assistance with DNA sequencing. This research is 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. O. K. is supported by an NIH predoctoral training fellowship from the National Institute of General Medical Sciences, NRSA Award 1 T32 GM 08346-01.
Funding AgencyGrant Number
Department of Energy (DOE)UNSPECIFIED
NIH Predoctoral Fellowship1 T32 GM 08346-01
Subject Keywords:directed evolution; DNA shuffling; random mutagenesis; para-nitrobenzyl esterase
Issue or Number:3
Record Number:CaltechAUTHORS:20180822-090200824
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Official Citation:Jeffrey C Moore, Hua-Ming Jin, Olga Kuchner, Frances H Arnold, Strategies for the in vitro evolution of protein function: enzyme evolution by random recombination of improved sequences11Edited by J. Wells, Journal of Molecular Biology, Volume 272, Issue 3, 1997, Pages 336-347, ISSN 0022-2836, (
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:89013
Deposited By: Tony Diaz
Deposited On:22 Aug 2018 16:23
Last Modified:16 Nov 2021 00:31

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