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A combinatorial approach to hybrid enzymes independent of DNA homology

Nature Biotechnology volume 17, pages 1205–1209 (1999)Cite this article

Abstract

We present a methodology, termed incremental truncation for the creation of hybrid enzymes (ITCHY), that creates combinatorial fusion libraries between genes in a manner that is independent of DNA homology. We compared the ability of ITCHY and DNA shuffling to create interspecies fusion libraries between fragments of the Escherichia coli and human glycinamide ribonucleotide transformylase genes, which have only 50% identity on the DNA level. Sequencing of several randomly selected positives from each library illustrated that ITCHY identified a more diverse set of active fusion points including those in regions of nonhomology and those with crossover points that diverged from the sequence alignment. Furthermore, some of the hybrids found by ITCHY that were fused at nonhomologous locations had activities that were greater than or equal to the activity of the hybrids found by DNA shuffling.

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Figure 1: ITCHY combinatorial protein engineering.
Figure 2: Size distribution of libraries.
Figure 3: Fusion points of active PurN–GART hybrids relative to the alignment of PurN and GART.
Figure 4: Fusion points of active PurN–GART hybrids mapped onto the structure of PurN31.

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Acknowledgements

We thank A.E. Nixon, C.P. Scott, and S.M. Firestine for many helpful discussions, and L.T. Gooljarsingh for purified GARS–AIRS–GART. This work was supported in part by National Institutes of Health Grant GM24129 (S.J.B.) and National Institutes of Health Postdoctoral Fellowship GM18560 (M.O.).

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  1. Department of Chemistry, The Pennsylvania State University, 414 Wartik Laboratory, University Park, 16802, PA

    Marc Ostermeier, Jae Hoon Shim & Stephen J. Benkovic

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  1. Marc Ostermeier
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Correspondence to Stephen J. Benkovic.

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Ostermeier, M., Shim, J. & Benkovic, S. A combinatorial approach to hybrid enzymes independent of DNA homology . Nat Biotechnol 17, 1205–1209 (1999). https://doi.org/10.1038/70754

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