A novel high-throughput (HTP) cloning strategy for site-directed designed chimeragenesis and mutation using the Gateway cloning system |
| |
Authors: | Suzuki Yasuhiro Kagawa Naoko Fujino Toru Sumiya Tsuyoshi Andoh Taichi Ishikawa Kumiko Kimura Rie Kemmochi Kiyokazu Ohta Tsutomu Tanaka Shigeo |
| |
Institution: | Primer Production Department, Invitrogen Corporation, Yokohama Kanazawa High-Tech Center Techno-Core Building.4F 1-1-1 Fukuura, Kanazawa, Yokohama, Kanagawa, 236-0004 Japan. |
| |
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. |
| |
Keywords: | |
本文献已被 PubMed 等数据库收录! |
|