Abstract
In vitro protein evolution is an efficient approach to study the structure and function of a protein, or to enhance its industrial utility (1). One round of evolution consists of random mutation of a protein-coding gene, expression the resulting library in a population of micro-organisms, and high-throughput screening or selection of clones that most strongly exhibit a desired phenotype (“winners”). After many rounds, mutations that confer the phenotype accumulate on a single allele, e.g., the authors have isolated an octuple mutant of the Escherichia coli β-glucuronidase with catalytic activity resistant to roughly 80-fold higher concentrations of glutaraldehyde than that of the wild-type enzyme (2). Here we describe a variation of the mutagenic polymerase chain reaction (PCR) (3,4) is described. The advantages of this method over other random mutagenesis techniques are explained.
Keywords
- Polymerase Chain Reaction
- Random Mutation
- Purify Polymerase Chain Reaction Product
- Shrimp Alkaline Phosphatase
- Qiaquick Polymerase Chain Reaction Purification
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.
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References
Sutherland, J. D. (2000) Evolutionary optimisation of enzymes. Curr. Opin. Chem. Biol. 4, 263–269.
Matsumura, I., Wallingford, J. B., Surana, N. K., Vize, P. D., and Ellington, A. D. (1999) Directed evolution of the surface chemistry of the reporter enzyme beta-glucuronidase. Nat. Biotechnol. 17, 696–701.
Cadwell, R. C. and Joyce, G. F. (1992) Randomization of genes by PCR mutagen-esis. PCR Methods Appl. 2, 28–33.
Cadwell, R. C. and Joyce, G. F. (1994) Mutagenic PCR. PCR Methods Appl. 3, S136–S140.
Zaccolo, M., Williams, D. M., Brown, D. M., and Gherardi, E. (1996) An approach to random mutagenesis of DNA using mixtures of triphosphate derivatives of nucleoside analogues. J. Mol. Biol. 255, 589–603.
Greener, A. and Callahan, M. (1994) XL 1-Red: a highly efficient random mutagenesis strain. Strategies (Stratagene) 7, 32–34.
Martinez, M. A., Pezo, V., Marliere, P., and Wain-Hobson, S. (1996) Exploring the functional robustness of an enzyme by in vitro evolution. EMBO J. 15, 1203–1210.
Black, M. E., Newcomb, T. G., Wilson, H. M., and Loeb, L. A. (1996) Creation of drug-specific herpes simplex virus type 1 thymidine kinase mutants for gene therapy. Proc. Natl. Acad. Sci. USA 93, 3525–3529.
Stemmer, W. P. (1994) DNA shuffling by random fragmentation and reassembly: in vitro recombination for molecular evolution. Proc. Natl. Acad. Sci. USA 91, 10,747–10,751.
Crameri, A., Raillard, S. A., Bermudez, E., and Stemmer, W. P. (1998) DNA shuffling of a family of genes from diverse species accelerates directed evolution. Nature 391, 288–291.
Fromant, M., Blanquet, S., and Plateau, P. (1995) Direct random mutagenesis of gene-sized DNA fragments using polymerase chain reaction. Anal. Biochem. 224, 347–353.
Matsumura, I. and Ellington, A. D. (1999) In vitro evolution of thermostable p53 variants. Protein Sci. 8, 731–740.
Miyazaki, K. and Arnold, F. H. (1999) Exploring nonnatural evolutionary pathways by saturation mutagenesis: rapid improvement of protein function. J. Mol. Evol. 49, 716–720.
Crameri, A., Whitehorn, E. A., Tate, E., and Stemmer, W. P. (1996) Improved green fluorescent protein by molecular evolution using DNA shuffling. Nat. Biotechnol. 14, 315–319.
Crameri, A., Dawes, G., Rodriguez, Jr., E., Silver, S., and Stemmer, W. P. (1997) Molecular evolution of an arsenate detoxification pathway by DNA shuffling. Nat. Biotechnol. 15, 436–438.
Stemmer, W. P. (1994) Rapid evolution of a protein in vitro by DNA shuffling. Nature 370, 389–391.
Zhang, J. H., Dawes, G., and Stemmer, W. P. (1997) Directed evolution of a fucosidase from a galactosidase by DNA shuffling and screening. Proc. Natl. Acad. Sci. USA 94, 4504–4509.
Zhao, H. and Arnold, F. H. (1997) Optimization of DNA shuffling for high fidelity recombination. Nucleic Acids Res. 25, 1307–1308.
King, P. V. and Blakesley, R. W. (1986) Optimizing DNA ligations for transformation. Focus (Gibco-BRL) 8, 1–3.
Inoue, H., Nojima, H., and Okayama, H. (1990) High efficiency transformation of Escherichia coli with plasmids. Gene 96, 23–28.
Beckman, R. A., Mildvan, A. S., and Loeb, L. A. (1985) On the fidelity of DNA replication: manganese mutagenesis in vitro. Biochemistry 24, 5810–5817.
Wybranietz, W. A. and Lauer, U. (1998) Distinct combination of purification methods dramatically improves cohesive-end subcloning of PCR products. Biotechniques 24, 578–580.
Thomas, M. R. (1994) Simple, effective cleanup of DNA ligation reactions prior to electro-transformation of E. coli. Biotechniques 16, 988–990.
Dower, W. J., Miller, J. F., and Ragsdale, C. W. (1988) High efficiency transformation of E. coli by high voltage electroporation. Nucleic Acids Res. 16, 6127–6145.
Moore, J. C. and Arnold, F. H. (1996) Directed evolution of a para-nitrobenzyl esterase for aqueous-organic solvents. Nat. Biotechnol. 14, 458–467.
Zaccolo, M. and Gherardi, E. (1999) The effect of high-frequency random mutagenesis on in vitro protein evolution: a study on TEM-1 beta-lactamase. J. Mol. Biol. 285, 775–783.
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Matsumura, I., Ellington, A.D. (2002). Mutagenic Polymerase Chain Reaction of Protein-Coding Genes for In Vitro Evolution. In: Braman, J. (eds) In Vitro Mutagenesis Protocols. Methods in Molecular Biology™, vol 182. Humana Press, Totowa, NJ. https://doi.org/10.1385/1-59259-194-9:259
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DOI: https://doi.org/10.1385/1-59259-194-9:259
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-0-89603-910-0
Online ISBN: 978-1-59259-194-7
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