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Random Mutagenesis for Protein Breeding

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Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 182))

Abstract

Mutagenesis is an essential process in evolution, generating an array of genetically unique organisms from which natural selection selects the best-suited to the immediate environment. Compatible and favorable mutations quickly converge; because offspring may inherit distinct advantageous mutations from both parents, and thus dramatically increase their survival rate. Parental crossing results in the accumulation of advantageous mutations in offspring. Evolutionary adaptation has allowed organisms to progressively specialize, and colonize, even in the most extreme of habitats.

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References

  1. Enoru-Eta, J., Gigot, D., Thia-Toong, T. L., Glansdorff, N., and Charlier, D. (2000) Purification and characterization of Sa-lrp, a DNA-binding protein from the extreme thermoacidophilic archaeon Sulfolobus acidocaldarius homologous to the bacterial global transcriptional regulator Lrp. J. Bacteriol. 182, 3661–3672.

    Article  PubMed  CAS  Google Scholar 

  2. Irgens, R. L., Gosink, J. J., and Staley, J. T. (1996) Polaromonas vacuolata gen. nov., sp. nov., a psychrophilic, marine, gas vacuolate bacterium from Antarctica. Int. J. Syst. Bacteriol. 46, 822–826.

    Article  PubMed  CAS  Google Scholar 

  3. Schmidt-Dannert, C. and Arnold, F. H. (1999) Directed evolution of industrial enzymes. Trends Biotechnol. 17, 135,136.

    Article  Google Scholar 

  4. Arnold, F. H. and Moore, J. C. (1997) Optimizing industrial enzymes by directed evolution. Adv. Biochem. Eng. Biotechnol. 58, 1–14.

    PubMed  CAS  Google Scholar 

  5. Marrs, B., Delagrave, S., and Murphy, D. (1999) Novel approaches for discovering industrial enzymes. Curr. Opin. Microbiol. 2, 241–245.

    Article  PubMed  CAS  Google Scholar 

  6. Buckland, B. C., Robinson, D. K., and Chartrain, M. (2000) Biocatalysis for pharmaceuticals-status and prospects for a key technology. Metab. Eng. 2, 42–48.

    Article  PubMed  CAS  Google Scholar 

  7. Wackett, L. P. (1998) Directed evolution of new enzymes and pathways for environmental biocatalysis. Ann. NY Ac ad. Sci. 864, 142–152.

    Article  CAS  Google Scholar 

  8. Jaeger, K. E. and Reetz, M. T. (2000) Directed evolution of enantioselective enzymes for organic chemistry. Curr. Opin. Chem. Biol. 4, 68–73.

    Article  PubMed  CAS  Google Scholar 

  9. 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.

    Article  PubMed  CAS  Google Scholar 

  10. MBI International. (2000) Extremophile Culture Collection. cr]2002 Humana Press Inc.http://www.mbi.org/extremophileculturecollec.htm.

  11. Kazlauskas, R. J. (2000) Molecular modeling and biocatalysis: explanations, predictions, limitations, and opportunities. Curr. Opin. Chem. Biol. 4, 81–88.

    Article  PubMed  CAS  Google Scholar 

  12. Moore, J. C., Jin, H. M., Kuchner, O., and Arnold, F. H. (1997) Strategies for the in vitro evolution of protein function: enzyme evolution by random recombination of improved sequences. J. Mol. Biol. 272, 336–347.

    Article  PubMed  CAS  Google Scholar 

  13. You, L. and Arnold, F. H. (1996) Directed evolution of subtilisin E in Bacillus subtilis to enhance total activity in aqueous dimethylformamide. Protein Eng. 9, 77–83.

    Article  PubMed  CAS  Google Scholar 

  14. Moore, J. C. and Arnold, F. H. (1996) Directed evolution of a para-nitrobenzyl esterase for aqueous-organic solvents. Nat. Biotechnol. 14, 458–467.

    Article  PubMed  CAS  Google Scholar 

  15. Kuchner, O. and Arnold, F. H. (1997) Directed evolution of enzyme catalysts. Trends Biotechnol. 15, 523–530.

    Article  PubMed  CAS  Google Scholar 

  16. Lanio, T., Jeltsch, A., and Pingoud, A. (2000) On the possibilities and limitations of rational protein design to expand the specificity of restriction enzymes: a case study employing EcoRV as the target. Protein Eng. 13, 275–281.

    Article  PubMed  CAS  Google Scholar 

  17. Liebeton, K., Zonta, A., Schimossek, K., Nardini, M., Lang, D., Dijkstra, B. W., et al. (2000) Directed evolution of an enantioselective lipase. Chem. Biol. 7, 709–718.

    Article  PubMed  CAS  Google Scholar 

  18. Reetz, M. T. and Jaeger, K. E. (2000) Enantioselective enzymes for organic synthesis created by directed evolution. Chemistry 6, 407–412.

    Article  PubMed  CAS  Google Scholar 

  19. Skandalis, A., Encell, L. P., and Loeb, L. A. (1997) Creating novel enzymes by applied molecular evolution. Chem. Biol. 4, 889–898.

    Article  PubMed  CAS  Google Scholar 

  20. Petrounia, I. P. and Arnold, F. H. (2000) Designed evolution of enzymatic properties. Curr. Opin. Biotechnol. 11, 325–330.

    Article  PubMed  CAS  Google Scholar 

  21. 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.

    Article  PubMed  CAS  Google Scholar 

  22. Kikuchi, M., Ohnishi, K., and Harayama, S. (2000) An effective family shuffling method using single-stranded DNA. Gene 243, 133–137.

    Article  PubMed  CAS  Google Scholar 

  23. Harayama, S. (1998) Artificial evolution by DNA shuffling. Trends Biotechnol. 16, 76–82.

    Article  PubMed  CAS  Google Scholar 

  24. Zhao, H. and Arnold, F. H. (1997) Optimization of DNA shuffling for high fidelity recombination. Nucleic Acids Res. 25, 1307,1308.

    Google Scholar 

  25. Lanio, T., Jeltsch, A., and Pingoud, A. (2000) Automated purification of His6-tagged proteins allows exhaustive screening of libraries generated by random mutagenesis. Biotechniques 29, 338–342.

    PubMed  CAS  Google Scholar 

  26. 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.

    Article  PubMed  CAS  Google Scholar 

  27. Stassen, A. P., Zaman, G. J., van Deursen, J. M., Schoenmakers, J. G., and Konings, R. N. (1992) Selection and characterization of randomly produced mutants of gene V protein of bacteriophage M13. Eur. J. Biochem. 204, 1003,1004.

    Article  Google Scholar 

  28. 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.

    Article  PubMed  CAS  Google Scholar 

  29. Matsuura, T., Miyai, K., Trakulnaleamsai, S., Yomo, T., Shima, Y., Miki, S., et al. (1999) Evolutionary molecular engineering by random elongation mutagenesis. Nat. Biotechnol. 17, 58–61.

    Article  PubMed  CAS  Google Scholar 

  30. Shao, Z., Zhao, H., Giver, L., and Arnold, F. H. (1998) Random-priming in vitro recombination: an effective tool for directed evolution. Nucleic Acids Res. 26, 681–683.

    Article  PubMed  CAS  Google Scholar 

  31. Cadwell, R. C. and Joyce, G. F. (1992) Randomization of genes by PCR mutagenesis. PCR Methods Appl. 2, 28–33.

    Article  PubMed  CAS  Google Scholar 

  32. Lin-Goerke, J. L., Robbins, D. J., and Burczak, J. D. (1997) PCR-based random mutagenesis using manganese and reduced dNTP concentration. Biotechniques 23, 409–412.

    PubMed  CAS  Google Scholar 

  33. Spee, J. H., de Vos, W. M., and Kuipers, O. P. (1993) Efficient random mutagenesis method with adjustable mutation frequency by use of PCR and dITP. Nucleic Acids Res. 21, 777,778.

    Article  Google Scholar 

  34. Schopf, J. W. and Packer, B. M. (1987) Early Archean (3.3-billion to 3.5-billion-year-old) microfossils from Warrawoona Group, Australia. Science 237, 70–73.

    Article  PubMed  CAS  Google Scholar 

  35. 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.

    Article  PubMed  CAS  Google Scholar 

  36. Xu, H., Petersen, E. I., Petersen, S. B., and El-Gewely, M. R. (1999) Random mutagenesis libraries: optimization and simplification by PCR. Biotechniques 27, 1102–1104,1106,1108.

    PubMed  CAS  Google Scholar 

  37. Copeland, W. C., Lam, N. K., and Wang, T. S. (1993) Fidelity studies of the human DNA polymerase alpha. The most conserved region among alpha-like DNA polymerases is responsible for metal-induced infidelity in DNA synthesis.J. Biol. Chem. 268, 11,041–11,049.

    PubMed  CAS  Google Scholar 

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Fenton, C., Xu, H., Petersen, E.I., Petersen, S.B., El-Gewely, M.R. (2002). Random Mutagenesis for Protein Breeding. In: Braman, J. (eds) In Vitro Mutagenesis Protocols. Methods in Molecular Biology™, vol 182. Humana Press, Totowa, NJ. https://doi.org/10.1385/1-59259-194-9:231

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  • DOI: https://doi.org/10.1385/1-59259-194-9:231

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-0-89603-910-0

  • Online ISBN: 978-1-59259-194-7

  • eBook Packages: Springer Protocols

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