A combinatorial approach to hybrid enzymes independent of DNA homology

Marc Ostermeier, Jae Hoon Shim, Stephen J. Benkovic

Research output: Contribution to journalArticlepeer-review

202 Scopus citations

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 coil 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.

Original languageEnglish (US)
Pages (from-to)1205-1209
Number of pages5
JournalNature Biotechnology
Volume17
Issue number12
DOIs
StatePublished - 1999

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology
  • Molecular Medicine
  • Biomedical Engineering

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