A novel high-throughput (HTP) cloning strategy for site-directed designed chimeragenesis and mutation using the Gateway cloning system Export

@article{suzuki05, abstract = {There is an increasing demand for easy, high-throughput (HTP) methods for protein engineering to support advances in the development of structural biology, bioinformatics and drug design. Here, we describe an N- and C-terminal cloning method utilizing Gateway cloning technology that we have adopted for chimeric and mutant genes production as well as domain shuffling. This method involves only three steps: PCR, in vitro recombination and transformation. All three processes consist of simple handling, mixing and incubation steps. We have characterized this novel HTP method on 96 targets with >90\% success. Here, we also discuss an N- and C-terminal cloning method for domain shuffling and a combination of mutation and chimeragenesis with two types of plasmid vectors.}, author = {Suzuki, Yasuhiro and Kagawa, Naoko and Fujino, Toru and Sumiya, Tsuyoshi and Andoh, Taichi and Ishikawa, Kumiko and Kimura, Rie and Kemmochi, Kiyokazu and Ohta, Tsutomu and Tanaka, Shigeo}, citeulike-article-id = {1570744}, doi = {doi:10.1093/nar/gni103}, journal = {Nucleic Acids Research}, month = {July}, number = {12}, posted-at = {2007-08-16 21:27:05}, priority = {0}, title = {A novel high-throughput ({HTP}) cloning strategy for site-directed designed chimeragenesis and mutation using the Gateway cloning system}, volume = {33}, year = {2005} }

TY - JOUR ID - suzuki05 L3 - citeulike-article-id:1570744 N2 - There is an increasing demand for easy, high-throughput (HTP) methods for protein engineering to support advances in the development of structural biology, bioinformatics and drug design. Here, we describe an N- and C-terminal cloning method utilizing Gateway cloning technology that we have adopted for chimeric and mutant genes production as well as domain shuffling. This method involves only three steps: PCR, in vitro recombination and transformation. All three processes consist of simple handling, mixing and incubation steps. We have characterized this novel HTP method on 96 targets with >90\% success. Here, we also discuss an N- and C-terminal cloning method for domain shuffling and a combination of mutation and chimeragenesis with two types of plasmid vectors. IS - 12 JF - Nucleic Acids Research TI - A novel high-throughput ({HTP}) cloning strategy for site-directed designed chimeragenesis and mutation using the Gateway cloning system VL - 33 AU - Suzuki, Yasuhiro AU - Kagawa, Naoko AU - Fujino, Toru AU - Sumiya, Tsuyoshi AU - Andoh, Taichi AU - Ishikawa, Kumiko AU - Kimura, Rie AU - Kemmochi, Kiyokazu AU - Ohta, Tsutomu AU - Tanaka, Shigeo PY - 2005/July// ER -