Abstract
Directed evolution by sequential cycles of random mutagenesis and screening has proven to be useful for producing new or improved enzyme properties (1,2). The first step is construction of a mutant library, usually accomplished by random point mutagenesis with error-prone PCR (3) or by DNA shuffling (recombination) (4,5). The second, and most critical step is finding the desired mutants by screening or selection of the libraries. Screens for enzyme activity usually operate via detection of optical absorption or fluorescence. Some screens are performed directly in agar plates, where changes of color are observed by direct visualization (6,7) or by digital image analysis (8). However, most screens involve transferring individual colonies to multi-well microplates containing culture medium, growing the cells until stationary phase, and inducing protein expression. If the target protein is intracellular, a cell lysis step has to be carried out to release the cellular contents. Finally, enzymatic activity is assayed with a microplate reader spectrophotometer. For many enzymes, thermal stability (9) is conveniently assayed by making a replica of the library and measuring the ratio of the activity after heating at defined temperature and time (residual activity, RA) to the activity measured before the heating (initial activity, IA) (see Chapter 11).
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References
Arnold, F. H., Wintrode, P., Miyazaki, K., and Gershenson, A. (2001) How enzymes adapt: lessons from directed evolution. Trends Biochem. Sci. 26, 100–106.
Arnold, F. H. (2001) Combinatorial and computational challenges for biocatalyst design. Nature 409, 253–257
Cadwell, R. C. and Joyce, G. F. (1992) Randomization of genes by PCR mutagenesis. PCR Methods Appl. 2, 28–33.
Stemmer, W. P. (1994) Rapid evolution of a protein in vitro by DNA shuffling. Nature 370, 389–391
Zhao, H., Giver, L., Shao, Z., Affholter, J. A, and Arnold, F. H. (1998) Molecular evolution by staggered extension process (StEP) in vitro recombination. Nat. Biotechnol. 16, 258–261.
Rellos, P. and Scopes, R. K. (1994) Polymerase chain reaction-based random mutagenesis: production and characterization of thermostable mutants of Zymomonas mobilis alcohol dehydrogenase-2. Protein Expr. Purif. 5, 270–277.
Lopez-Camacho, C., Salgado, J., Lequerica, J. L., et al. (1996) Amino acid substitutions enhancing thermostability of Bacillus polymyxa β-glucosidase A. Biochem. J. 314, 833–838.
Joo, H., Arisawa, A., Lin, Z., and Arnold, F. H. (1999) A high-throughput digital imaging screen for the discovery and directed evolution of oxygenases. Chem. Biol. 6, 699–706.
Gershenson, A. and Arnold, F. H. (2000) Enzyme stabilization by directed evolution. Genetic Eng. 22, 55–76.
Kuchner, O. and Arnold, F. H. (1997) Directed evolution of enzyme catalysts. Trends Biotechnol. 15, 523–530.
Shafikhani, S., Siegel, R.A., Ferrari, E., and Schellenberg, V. (1997) Generation of large libraries of random mutants in Bacillus subtilis by PCR-based plasmid multimerization. Biotechniques 23, 304–310.
Makrides, S. C. (1996) Strategies for achieving high level expression of genes in Escherichia coli. Microbiol. Rev. 60, 512–538.
Fridjonsson, O., Watzlawick, H., and Mattes, R. (2002) Thermoadaptation of α-galactosidase AgaB1 in Thermus thermophilus. J. Bacteriol. 184, 3385–3391.
You, L. and Arnold, F. H. (1994) Directed evolution of subtilisin E in B. subtilis to enhance total activity in aqueous dimethylformamide. Protein Eng. 9, 77–83.
Jonasson, P., Liljeqvist, S., Nygren, P-A., and Stahl, S. (2002) Genetic design for facilitated production and recovery of recombinant proteins in Escherichia coli. Biotechnol. Appl. Biochem. 35, 91–105.
Wintrode, P. L., Miyazaki, K., and Arnold, F. H. (2001) Patterns of adaptation in a laboratory evolved thermophilic enzyme. Biochem. Biophys. Acta. 1549, 1–8.
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© 2003 Humana Press Inc., Totowa, NJ
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Salazar, O., Sun, L. (2003). Evaluating a Screen and Analysis of Mutant Libraries. In: Arnold, F.H., Georgiou, G. (eds) Directed Enzyme Evolution. Methods in Molecular Biology™, vol 230. Humana Press. https://doi.org/10.1385/1-59259-396-8:85
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DOI: https://doi.org/10.1385/1-59259-396-8:85
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