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| Why high-error-rate random mutagenesis libraries are enriched in functional and improved proteins |
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| Author(s): Drummond DA, Iverson BL, Georgiou G, Arnold FH |
| Source: JOURNAL OF MOLECULAR BIOLOGY Volume: 350 Issue: 4 Pages: 806-816 Published: JUL 22 2005 |
| Times Cited: 38 References: 25 |
| Abstract: 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 errorprone 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. (c) 2005 Elsevier Ltd. All rights reserved. |
| Document Type: Article |
| Language: English |
| Reprint Address: Arnold, FH (reprint author), CALTECH, Div Chem & Chem Engn, Mail Code 210-41, Pasadena, CA 91125 USA |
Addresses:
1. CALTECH, Div Chem & Chem Engn, Pasadena, CA 91125 USA
2. CALTECH, Program Computat & Neural Syst, Pasadena, CA 91125 USA
3. Univ Texas, Dept Chem & Biochem, Austin, TX 78712 USA
4. Univ Texas, Dept Chem Engn, Austin, TX 78712 USA |
| Publisher: ACADEMIC PRESS LTD ELSEVIER SCIENCE LTD, 24-28 OVAL RD, LONDON NW1 7DX, ENGLAND |
| Subject Category: Biochemistry & Molecular Biology |
| IDS Number: 944RG |
| ISSN: 0022-2836 |
| DOI: 10.1016/j.jmb.2005.05.023 |
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