TY - JOUR
T1 - A combinatorial approach to hybrid enzymes independent of DNA homology
AU - Ostermeier, Marc
AU - Shim, Jae Hoon
AU - Benkovic, Stephen J.
N1 - Funding Information:
We thank A.E. Nixon, C.P. Scott, and S.M. Firestine for many helpful discussions, and L.T. Gooljarsingh for purified GARS-AIRS-GART. This work was supported in part by National Institutes of Health Grant GM24129 (S.J.B.) and National Institutes of Health Postdoctoral Fellowship GM18560 (M.O.).
PY - 1999
Y1 - 1999
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=17444363724&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=17444363724&partnerID=8YFLogxK
U2 - 10.1038/70754
DO - 10.1038/70754
M3 - Article
C2 - 10585719
AN - SCOPUS:17444363724
SN - 1087-0156
VL - 17
SP - 1205
EP - 1209
JO - Nature Biotechnology
JF - Nature Biotechnology
IS - 12
ER -