Research Article | Published:

Molecular evolution by staggered extension process (StEP) in vitro recombination

Nature Biotechnology volume 16, pages 258261 (1998) | Download Citation

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Abstract

We have developed a simple and efficient method for in vitro mutagenesis and recombination of polynu-cleotide sequences. The staggered extension process (StEP) consists of priming the template sequence(s) followed by repeated cycles of denaturation and extremely abbreviated annealing/polymerase-catalyzed extension. In each cycle the growing fragments anneal to different templates based on sequence complementarity and extend further. This is repeated until full-length sequences form. Due to template switching, most of the polynucleotides contain sequence information from different parental sequences. The method is demonstrated by the recombination of two genes encoding thermostable subtilisins carrying two phenotypic markers separated by 113 base pairs and eight other point mutation markers. To demonstrate its utility for directed evolution, we have used StEP to recombine a set of five thermostabilized subtilisin E variants identified during a single round of error-prone PCR mutagenesis and screening. Screening the StEP-recombined library yielded an enzyme whose half-life at 65°C is 50 times that of wild-type subtilisin E.

References

  1. 1.

    1987. Genetic recombination. Sci. Am. 256:1–101.

  2. 2.

    and 1988. Genetic recombination. American Society of Microbiology, Washington, DC.

  3. 3.

    1992. Genetic algorithms. Sci. Am. 267:66–72.

  4. 4.

    1992. Adaptation in natural and artificial systems. 2nd ed. MIT Press, Cambridge, MA,

  5. 5.

    1993. Genetic algorithms: Principles of natural selection applied to computation. Science 261:872–878.

  6. 6.

    and 1997. Directed evolution of enzyme catalysts. Trends Biotech. 15:523–530.

  7. 7.

    1994. Rapid evolution of a protein in vitro by DNA shuffling. Nature 370:389–391.

  8. 8.

    1994. DNA shuffling by random fragmentation and reassembly - In vitro recombination for molecular evolution. Proc. NaU. Acad. Sci. USA 91:10747–10751.

  9. 9.

    and 1997. Functional and nonfunctional mutations distinguished by random recombination of homologous genes. Proc. Natl. Acad. Sci. USA 94:7997–8000.

  10. 10.

    , , and 1998. Random-priming in vitro recombination: an effective tool for directed evolution. Nucl. Acids Res. 26:681–683.

  11. 11.

    , , and 1997. Generation of large libraries of random mutants in Bacillus subtilis by PCR-based plasmid multimerization. Biotechniques23:304–310.

  12. 12.

    and 1997. Optimization of DNA shuffling for high-fidelity recombination. Nucl. Acids Res.25:1307–1308.

  13. 13.

    , , , , , et al. 1998. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239:487–491.

  14. 14.

    , , , ,, 1997. HLA class II allelic variation and susceptibility to pemphigus vulgaris. Proc. Natl. Acad. Sci. USA 85:3504–3508.

  15. 15.

    , and 1998. Sequence analysis of the HLA-DRB and HLA-DQB loci from 3 pemphigus vulgaris patients. Hum. Immunol. 22:61–69.

  16. 16.

    and 1997. Recombinant DNA sequences generated by PCR amplification. Mol. Biol. Evol. 14:592–593.

  17. 17.

    1989. Taq DNA polymerase, p. 18 in PCR technology. Ehrlich, H.A. (ed.). Stockton Press, New York.

  18. 18.

    and 1996. Directed evolution of a para-nitrobenzyl esterase. Bio/Technology 14:458–467.

  19. 19.

    , , and 1997. Strategies for the in vitro evolution of protein function: enzyme evolution by random recombination of improved sequences. J. Mol Biol. 272:336–347.

  20. 20.

    , , and 1997. Homologous recombination occurs in a distinct retroviral subpopulation and exhibits high negative interference. J. Virol. 71:6028–6036.

  21. 21.

    and 1994. Mutagenic PCR. PCP Methods and Applications 2:28–33.

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Author information

Affiliations

  1. Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125

    • Huimin Zhao
    • , Lori Giver
    • , Zhixin Shao
    •  & Frances H. Arnold
  2. Materials R&D-Biocatalysis, The Dow Chemical Company, 1707 Building, Midland, MI 48674.

    • Joseph A. Affholter

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Correspondence to Frances H. Arnold.

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https://doi.org/10.1038/nbt0398-258

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