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Synthetic shuffling expands functional protein diversity by allowing amino acids to recombine independently

Nature Biotechnology volume 20, pages 1251–1255 (2002)Cite this article

Abstract

We describe synthetic shuffling, an evolutionary protein engineering technology in which every amino acid from a set of parents is allowed to recombine independently of every other amino acid. With the use of degenerate oligonucleotides, synthetic shuffling provides a direct route from database sequence information to functional libraries. Physical starting genes are unnecessary, and additional design criteria such as optimal codon usage or known beneficial mutations can also be incorporated. We performed synthetic shuffling of 15 subtilisin genes and obtained active and highly chimeric enzymes with desirable combinations of properties that we did not obtain by other directed-evolution methods.

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Figure 1: Oligonucleotide design strategy for synthetic shuffling of subtilisins.
Figure 2: Comparative phylogenies of synthetic shuffled and fragmentation-shuffled library members.
Figure 3: Activity of synthetic and fragmentation-based libraries.
Figure 4: Sequences of variants with new combinations of physical properties.
Figure 5: A six–amino acid region of subtilisin recombined by synthetic shuffling.

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Acknowledgements

We thank Mark Welch for screening and automation assistance, Andreas Crameri and Pim Stemmer for advice on library design and assembly parameters, Troy Obrero and Walker Lutringer for DNA sequencing, Ajoy Roy for statistical advice, and Allan Svendsen and Bo Hammer for critically reading the manuscript.

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Authors and Affiliations

  1. Maxygen, 515 Galveston Drive, Redwood City, 94063, CA, USA

    Jon E. Ness, Seran Kim, Andrea Gottman, Rob Pak, Anke Krebber, Sridhar Govindarajan, Emily C. Mundorff & Jeremy Minshull

  2. Protein Design, Novozymes, Building 2C, Bagsværd, DK 2880, Denmark

    Torben V. Borchert

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  1. Jon E. Ness
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Ness, J., Kim, S., Gottman, A. et al. Synthetic shuffling expands functional protein diversity by allowing amino acids to recombine independently. Nat Biotechnol 20, 1251–1255 (2002). https://doi.org/10.1038/nbt754

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