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The PRESAT‐vector: Asymmetric T‐vector for high‐throughput screening of soluble protein domains for structural proteomics
Author(s) -
Goda Natsuko,
Tenno Takeshi,
Takasu Hirotoshi,
Hiroaki Hidekazu,
Shirakawa Masahiro
Publication year - 2004
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.03439004
Subject(s) - restriction enzyme , fusion protein , plasmid , expression vector , cloning (programming) , multiple cloning site , biology , transformation (genetics) , microbiology and biotechnology , complementary dna , vector (molecular biology) , cloning vector , restriction site , primer (cosmetics) , computational biology , molecular cloning , genetics , dna , recombinant dna , chemistry , gene , computer science , organic chemistry , programming language
A rapid unidirectional method for cloning PCR‐amplified cDNA fragments into virtually any fusion protein expression vector is described. The method, termed PRESAT‐vector cloning, is based on a T‐vector technique that does not require restriction endonuclease digestion of the PCR product. Subsequently, we applied a novel ORF selection method of the ligated plasmid products. This second step involves restriction endonuclease treatment that eliminates the plasmids containing an ORF in the wrong orientation prior to transformation into the bacterial host for further protein expression studies. To achieve this selection, we customized the 5′‐sequence of the “rear” PCR primer corresponding to the C terminus of the protein to be expressed. The colonies harbored only the ligated products of the desired orientation at >90% efficiency. This method is applied to a GST fusion expression system, and an HTS system for soluble proteins from an expression library was tested.