Abstract
The evolution of proteins is more difficult than the evolution of nucleic acids both in principle and in practice. While nucleic acid sequence space has a dimensionality of 4n, where n is the size of the nucleic acid pool (i.e., G, C, A, and T), protein sequence space has a dimensionality of 20n. Similarly, while nucleic acids can frequently be directly selected for function from a random sequence population, the corresponding methods for the directed evolution of proteins are generally not as robust, in part because of the larger sequence spaces that must be explored, and in part because protein selection requires a translation step that in turn often requires cellular transformation, an inherently inefficient procedure that limits library size. In addition, the requirement for expression of the protein library in a host places limits on the numbers and types of selections that can be performed. Selecting individual colonies on plates is not well-suited to truly high-throughput methods and generally limits library sizes to on the order of 105. Moreover, the complexity of cellular metabolism provides an almost limitless source of potential artifacts to confound the selection of a given phenotype. For example, attempts to evolve an antibiotic resistance element can be thwarted by the evolution of chromosomal resistance elements or by the evolution of plasmid copy number or promoter strength rather than protein efficiency (1,2). While there are frequently work-arounds for many of the artifacts that might be encountered, they nonetheless ultimately limit the phenotypes that can be selected.
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References
Normark, B. H. and Normark, S. (2002) Evolution and spread of antibiotic resistance. J. Intern. Med. 252, 91–106.
Mortlock, R. P. (1982) Metabolic acquisitions through laboratory selection. Annu. Rev. Microbiol. 36, 259–284.
Brisson, M., He, Y., Li, S., Yang, J. P., and Huang, L. (1999) A novel T7 RNA polymerase autogene for efficient cytoplasmic expression of target genes. Gene Ther. 6, 263–270.
Li, S., Brisson, M., He, Y., and Huang, L. (1997) Delivery of a PCR amplified DNA fragment into cells: a model for using synthetic genes for gene therapy. Gene Ther. 4, 449–454.
Walker, K., Xie, Y., Li, Y., et al. (2001) Cytoplasmic expression of ribozyme in zebrafish using a T7 autogene system. Curr. Issues Mol. Biol. 3, 1–6.
Ghadessy, F. J., Ong, J. L., and Holliger, P. (2001) Directed evolution of polymerase function by compartmentalized self-replication. Proc. Natl. Acad. Sci. USA 98, 4552–4557.
Dubendorff, J. W. and Studier, F. W. (1991) Controlling basal expression in an inducible T7 expression system by blocking the target T7 promoter with lac repressor. J. Mol. Biol. 219, 45–59.
Dubendorff, J. W. and Studier, F. W. (1991) Creation of a T7 autogene. Cloning and expression of the gene for bacteriophage T7 RNA polymerase under control of its cognate promoter. J. Mol. Biol. 219, 61–68.
Chelliserrykattil, J., Cai, G., and Ellington, A. D. (2001) A combined in vitro/in vivo selection for polymerases with novel promoter specificities. BMC Biotechnol. 1, 13.
Sarkar, P., Sengupta, D., Basu, S., and Maitra, U. (1985) Nucleotide sequence of a major class-III phage-T3 RNA-polymerase promoter located at 98.0% of phage-T3 genetic map. Gene 33, 351–355.
Adhya, S., Basu, S., Sarkar, P., and Maitra, U. (1981) Location, function, and nucleotide sequence of a promoter for bacteriophage T3 RNA polymerase. Proc. Natl. Acad. Sci. USA 78, 147–151.
Bailey, J. N., Klement, J. F., and McAllister, W. T. (1983) Relationship between promoter structure and template specificities exhibited by the bacteriophage T3 and T7 RNA polymerases. Proc. Natl. Acad. Sci. USA 80, 2814–2818.
Raskin, C. A., Diaz, G., Joho, K., and McAllister, W. T. (1992) Substitution of a single bacteriophage T3 residue in bacteriophage T7 RNA polymerase at position 748 results in a switch in promoter specificity. J. Mol. Biol. 228, 506–515.
Rong, M., He, B., McAllister, W. T., and Durbin, R. K. (1998) Promoter specificity determinants of T7 RNA polymerase. Proc. Natl. Acad. Sci. USA 95, 515–519.
Raskin, C. A., Diaz, G. A., and McAllister, W. T. (1993) T7 RNA polymerase mutants with altered promoter specificities. Proc. Natl. Acad. Sci. USA 90, 3147–3151.
Imburgio, D., Rong, M., Ma, K., and McAllister, W. T. (2000) Studies of promoter recognition and start site selection by T7 RNA polymerase using a comprehensive collection of promoter variants. Biochemistry 39, 10,419–10,430.
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.
Dower, W. J., Miller, J. F., and Ragsdale, C. W. (1988) High efficiency transformation of E. coli by high voltage electroporation. Nucl. Acids Res. 16, 6127–6145.
Thomas, M. R. (1994) Simple, effective cleanup of DNA ligation reactions prior to electro-transformation of E. coli. Biotechniques 16, 988–990.
Schmitz, A. and Galas, D. J. (1979) The interaction of RNA polymerase and lac repressor with the lac control region. Nucl. Acids Res. 6, 111–137.
Dunaway, M., Olson, J. S., Rosenberg, J. M., Kallai, O. B., Dickerson, R. E., and Matthews, K. S. (1980) Kinetic studies of inducer binding to lac repressor.operator complex. J. Biol. Chem. 255, 10,115–10,119.
Moffatt, B. A., and Studier, F. W. (1987) T7 lysozyme inhibits transcription by T7 RNA polymerase. Cell 49, 221–227.
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Chelliserrykattil, J., Ellington, A.D. (2003). Autogene Selections. In: Arnold, F.H., Georgiou, G. (eds) Directed Enzyme Evolution. Methods in Molecular Biology™, vol 230. Humana Press. https://doi.org/10.1385/1-59259-396-8:27
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DOI: https://doi.org/10.1385/1-59259-396-8:27
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