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

Nature Biotechnology volume 16, pages 258–261 (1998)Cite this article

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.

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Authors and 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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  1. Huimin Zhao
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  2. Lori Giver
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  3. Zhixin Shao
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  4. Joseph A. Affholter
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  5. Frances H. Arnold
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Correspondence to Frances H. Arnold.

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Zhao, H., Giver, L., Shao, Z. et al. Molecular evolution by staggered extension process (StEP) in vitro recombination. Nat Biotechnol 16, 258–261 (1998). https://doi.org/10.1038/nbt0398-258

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