POSITIVE SELECTION VECTORS TO GENERATE FUSED GENES FOR THE EXPRESSIONOF HIS-TAGGED PROTEINS

Citation
T. Vanreeth et al., POSITIVE SELECTION VECTORS TO GENERATE FUSED GENES FOR THE EXPRESSIONOF HIS-TAGGED PROTEINS, BioTechniques, 25(5), 1998, pp. 898-904
Citations number
13
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biology,"Biochemical Research Methods
Journal title
BioTechniques → ACNP
ISSN journal
0736-6205
Volume
25
Issue
5
Year of publication
1998
Pages
898 - 904
Database
ISI
SICI code
0736-6205(1998)25:5<898:PSVTGF>2.0.ZU;2-Y
Abstract
Epitope ragging simplifies detection, characterization and purificatio n of proteins. Gene fusion to combine the coding region of a well-char acterized epitope with the coding region for a protein of interest gen erally requires several subcloning steps. Alternatively, a PCR strateg y can be used to generate such a chimeric gene. In addition to irs sim plicity, this approach allows one to limit the size of the multiple cl oning sites present in conventional expression vectors, thus reducing the introduction of artifactual amino-acid sequences into the fused pr otein. In this communication, we describe new vectors that allow PCR c loning and selection of chimeric genes coding for N- or C-terminal His -tagged proteins. These vectors are based on the control of cell death CcdB direct selection technology and are well adapted to the cloning of blunt-ended PCR products that,were generated by using thermostable polymerases that provide proofreading activity.