A novel high-throughput (HTP) cloning strategy for site-directed designed chimeragenesis and mutation using the Gateway cloning system.
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.
Suzuki
Yasuhiro
Y
Primer Production Department, Invitrogen Corporation, Yokohama Kanazawa High-Tech Center Techno-Core Building.4F 1-1-1 Fukuura, Kanazawa, Yokohama, Kanagawa, 236-0004 Japan.
Kagawa
Naoko
N
Fujino
Toru
T
Sumiya
Tsuyoshi
T
Andoh
Taichi
T
Ishikawa
Kumiko
K
Kimura
Rie
R
Kemmochi
Kiyokazu
K
Ohta
Tsutomu
T
Tanaka
Shigeo
S
D003001:Cloning, Molecular / Q000379:methods*; D032621:DNA Shuffling; D004270:DNA, Circular / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000737:chemistry; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000235:genetics*; D011995:Recombination, Genetic; D012689:Sequence Homology, Nucleic Acid; D014169:Transformation, Bacterial
D004270:DNA, Circular; D011993:Recombinant Fusion Proteins