A combinatorial approach to hybrid enzymes independent of DNA homology

We present a methodology, termed incremental truncation for the creation of hybrid enzymes (ITCHY), that creates combinatorial fusion libraries betwee n genes in a manner that is independent of DNA homology. We compared the ab ility of ITCHY and DNA shuffling to create interspecies fusion libraries be tween fragments of the Escherichia coli and human glycinamide ribonucleotid e transformylase genes, which have only 50% identity on the DNA level. Sequ encing 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 diver ged from the sequence alignment. Furthermore, some of the hybrids found by ITCHY that were fused at nonhomologous locations had activities that were g reater than or equal to the activity of the hybrids found by DNA shuffling.