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Why High-error-rate Random Mutagenesis Libraries are Enriched in Functional and Improved Proteins

Drummond, D. Allan and Iverson, Brent L. and Georgiou, George and Arnold, Frances H. (2005) Why High-error-rate Random Mutagenesis Libraries are Enriched in Functional and Improved Proteins. Journal of Molecular Biology, 350 (4). pp. 806-816. ISSN 0022-2836. doi:10.1016/j.jmb.2005.05.023.

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The fraction of proteins that retain wild-type function after mutation has long been observed to decline exponentially as the average number of mutations per gene increases. Recently, several groups have used error-prone polymerase chain reactions (PCR) to generate libraries with 15 to 30 mutations per gene, on average, and have reported that orders of magnitude more proteins retain function than would be expected from the low-mutation-rate trend. Proteins with improved or novel function were isolated disproportionately from these high-error-rate libraries, leading to claims that high mutation rates unlock regions of sequence space that are enriched in positively coupled mutations. Here, we show experimentally that error-prone PCR produces a broader non-Poisson distribution of mutations consistent with a detailed model of PCR. As error rates increase, this distribution leads directly to the observed excesses in functional clones. We then show that while very low mutation rates result in many functional sequences, only a small number are unique. By contrast, very high mutation rates produce mostly unique sequences, but few retain function. Thus an optimal mutation rate exists that balances uniqueness and retention of function. Overall, high-error-rate mutagenesis libraries are enriched in improved sequences because they contain more unique, functional clones. Our findings demonstrate how optimal error-prone PCR mutation rates may be calculated, and indicate that “optimal” rates depend on both the protein and the mutagenesis protocol.

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Arnold, Frances H.0000-0002-4027-364X
Additional Information:© 2005 Elsevier Ltd. Received 18 February 2005, Revised 6 May 2005, Accepted 10 May 2005, Available online 25 May 2005. We thank G. Chen and R. Loo for creation and screening of the scFv libraries, J. D. Bloom for optimal mutation rate calculations, C. C. Adami for guidance, C. O. Wilke for lattice protein folding code and advice, and Z. -G. Wang for insightful comments on the manuscript. We are grateful for the critical input of two anonymous reviewers. D.A.D. acknowledges NIH National Research Service Award 5 T32 MH19138. This research is supported by Army Research Office Contract DAAD19-03-D-0004.
Funding AgencyGrant Number
NIH Predoctoral Fellowship5 T32 MH19138
Army Research Office (ARO)DAAD19-03-D-0004
Subject Keywords:mutagenesis; PCR; optimal mutation rate; Poisson distribution; neutrality
Issue or Number:4
Record Number:CaltechAUTHORS:20180821-161657424
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Official Citation:D. Allan Drummond, Brent L. Iverson, George Georgiou, Frances H. Arnold, Why High-error-rate Random Mutagenesis Libraries are Enriched in Functional and Improved Proteins, Journal of Molecular Biology, Volume 350, Issue 4, 2005, Pages 806-816, ISSN 0022-2836, (
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:89001
Deposited By: Tony Diaz
Deposited On:21 Aug 2018 23:26
Last Modified:16 Nov 2021 00:31

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