Abstract
Directed protein evolution is usually accomplished by generating random or targeted mutations in the protein coding sequence and screening the mutant proteins (library) for functional improvements. By targeting mutations to certain amino acids, we can map the enzyme active site, investigate mechanisms, and study structure-function relationships. With saturation mutagenesis, it is possible to create a library of mutants containing all possible mutations at one or more pre-determined target positions in a gene sequence.
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References
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.
Miyazaki, K., Wintrode, P. L., Grayling, R. A., Rubingh, D. N., and Arnold, F. H. (2000) Directed evolution study of temperature adaptation in a psychrophilic enzyme. J. Mol. Biol. 297, 1015–1026.
Morawski, B., Quan, S., and Arnold, F. H. (2001) Functional expression and stabilization of horseradish peroxidase by directed evolution in Saccharomyces cerevisiae. Biotechnol. Bioeng. 76, 99–107.
Sakamoto, T., Joern, J. M., Arisawa, A., and Arnold, F. H. (2001) Laboratory evolution of toluene dioxygenase to accept 4-picoline as a substrate. Appl. Environ. Microbiol. 67, 3882–3887.
May, O., Nguyen, P. T., and Arnold, F. H. (2000) Inverting enantioselectivity by directed evolution of hydantoinase for improved production of l-methionine. Nat. Biotechnol. 18, 317–320.
Babbitt, P. C. and Gerlt, J. A. (1997) Understanding enzyme superfamilies. Chemistry As the fundamental determinant in the evolution of new catalytic activities. J. Biol. Chem. 272, 30,591–30,594.
Horwitz, M. S. and Loeb, L. A. (1986) Promoters selected from random DNA sequences. Proc. Natl. Acad. Sci. USA 83, 7405–7409.
Whittle, E. and Shanklin, J. (2001) Engineering delta 9–16∶0-acyl carrier protein (ACP) desaturase specificity based on combinatorial saturation mutagenesis and logical redesign of the castor delta 9–18∶0-ACP desaturase. J. Biol. Chem. 276, 21,500–21,505.
Kegler-Ebo, D., Pollack, G., and DiMaio, D. (1996) Use of Codon Cassette Mutagenesis for Saturation Mutagenesis, in In Vitro Mutagenesis Protocols (Trower, M. K., ed.), Humana Press, Totowa, NJ, pp. 297–311.
Chiang, L. W. (1996) Saturation Mutagenesis by Mutagenic Oligonucleotide-Directed PCR Amplification (Mod-PCR), in In Vitro Mutagenesis Protocols (Trower, M. K., ed.), Humana Press, Totowa, NJ, pp. 311–321.
Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K., and Pease, L. R. (1989) Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77, 51–59.
Cline, J., Braman, J. C., and Hogrefe, H. H. (1996) PCR fidelity of Pfu DNA polymerase and other thermostable DNA polymerases. Nucleic Acids Res. 24, 3546–3551.
Reidhaar-Olson, J. F., Bowie, J. U., Breyer, R. M., et al. (1991) Random mutagenesis of protein sequences using oligonucleotide cassettes. Meth. Enzymol. 208, 564–586.
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© 2003 Humana Press Inc., Totowa, NJ
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Georgescu, R., Bandara, G., Sun, L. (2003). Saturation Mutagenesis. In: Arnold, F.H., Georgiou, G. (eds) Directed Evolution Library Creation. Methods in Molecular Biology™, vol 231. Humana Press. https://doi.org/10.1385/1-59259-395-X:75
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DOI: https://doi.org/10.1385/1-59259-395-X:75
Publisher Name: Humana Press
Print ISBN: 978-1-58829-285-8
Online ISBN: 978-1-59259-395-8
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