Incremental truncation as a strategy in the engineering of novel biocatalysts.
The application and success of combinatorial approaches to protein engineering problems have increased dramatically. However, current directed evolution strategies lack a combinatorial methodology for creating libraries of hybrid enzymes which lack high homology or for creating libraries of highly homologous genes with fusions at regions of non-identity. To create such hybrid enzyme libraries, we have developed a series of combinatorial approaches 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 engineering as well as protein folding, enzyme evolution, and the chemical synthesis of proteins. In addition, we are developing a methodology of DNA shuffling which is independent of DNA sequence homology.
Ostermeier
M
M
Department of Chemistry, The Pennsylvania State University, University Park 16802, USA.
Nixon
A E
AE
Benkovic
S J
SJ
D020816:Amino Acid Motifs; D002384:Catalysis; D020650:Combinatorial Chemistry Techniques; D019281:Dimerization; D004798:Enzymes / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005090:Exodeoxyribonucleases / Q000235:genetics / Q000378:metabolism; D015723:Gene Library; D005821:Genetic Techniques; D019151:Peptide Library; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism
D004798:Enzymes; D019151:Peptide Library; D011993:Recombinant Fusion Proteins; D005090:Exodeoxyribonucleases; C020133:exodeoxyribonuclease III