Construction of hybrid gene libraries involving the circular permutation of DNA

Marc Ostermeier, Stephen J. Benkovic

Research output: Contribution to journalArticle

11 Scopus citations

Abstract

The method of incremental truncation for the creation of hybrid enzymes (ITCHY) allows the creation of comprehensive fusion libraries between 5′ and 3′ fragments of two genes in a manner that is independent of DNA sequence homology. A methodology is presented for the creation of ITCHY libraries called circularly permuted ITCHY (CP-ITCHY) that allows the creation of ITCHY libraries in a manner that does not require extensive time point sampling. In addition, CP-ITCHY requires only a single vector and productively biases the library towards those fusions that are approximately the same size as the original genes. In the model system of creating fusions between fragments of the Escherichia coli and human glycinamide ribonucleotide transformylase genes, the CP-ITCHY libraries are shown to contain a diverse set of active fusions including those in regions of low-homology. In addition, a high percentage of active fusions were temperature-sensitive as they complemented an auxotrophic strain of Escherichia coli at 22°C but not at 37°C.

Original languageEnglish (US)
Pages (from-to)303-310
Number of pages8
JournalBiotechnology Letters
Volume23
Issue number4
DOIs
StatePublished - Feb 28 2001

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology

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