TY - JOUR
T1 - Construction of hybrid gene libraries involving the circular permutation of DNA
AU - Ostermeier, Marc
AU - Benkovic, Stephen J.
N1 - Funding Information:
We thank S. Lutz for many helpful discussions and for a critical reading of this manuscript and G. S. Baird for advice on circular permutation. This work was supported in part by National Institutes of Health Grant GM24129 (SJB).
PY - 2001
Y1 - 2001
N2 - 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.
AB - 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.
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U2 - 10.1023/A:1005642725295
DO - 10.1023/A:1005642725295
M3 - Article
AN - SCOPUS:0035108527
SN - 0141-5492
VL - 23
SP - 303
EP - 310
JO - Biotechnology Letters
JF - Biotechnology Letters
IS - 4
ER -