Incremental truncation as a strategy in the engineering of novel biocatalysts

The application and success of combinatorial approaches to protein engineer ing problems have increased dramatically. However, current directed evoluti on strategies lack a combinatorial methodology for creating libraries of hy brid enzymes which lack high homology or for creating libraries of highly h omologous genes with fusions at regions of non-identity. To create such hyb rid enzyme libraries, we have developed a series of combinatorial approache s that utilize the incremental truncation of genes, gene fragments or gene libraries. For incremental truncation, Exonuclease III is used to create a library of all possible single base-pair deletions of a given piece of DNA. Incremental truncation libraries (ITLs) have applications in protein engin eering as well as protein folding, enzyme evolution, and the chemical synth esis of proteins. In addition, we are developing a methodology of DNA shuff ling which is independent of DNA sequence homology. (C) 1999 Elsevier Scien ce Ltd. All rights reserved.