High-throughput T7 LIC vector for introducing C-terminal poly-histidine tags with variable lengths without extra sequences.
Protein Expr Purif
; 63(1): 58-61, 2009 Jan.
Article
en En
| MEDLINE
| ID: mdl-18824233
ABSTRACT
Immobilized metal ion affinity chromatography (IMAC) has become one of the most popular protein purification methods for recombinant proteins with a hexa-histidine tag (His-tag) placed at the C- or N-terminus of proteins. Nevertheless, there are always difficult proteins that show weak binding to the metal chelating resin and thus low purity. These difficulties are often overcome by increasing the His-tag to 8 or 10 histidines. Despite their success, there are only few expression vectors available to easily clone and test different His-tag lengths. Therefore, we have modified Escherichia coli T7 expression vector pET21a to accommodate ligation-independent cloning (LIC) that will allow easy and efficient parallel cloning of target genes with different His-tag lengths using a single insert. Unlike most LIC vectors available commercially, our vectors will not translate unwanted extra sequences by engineering the N-terminal linker to anneal before the open reading frame, and the C-terminal linker to anneal as a His-tag.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Proteínas Recombinantes de Fusión
/
Clonación Molecular
/
Bacteriófago T7
/
Vectores Genéticos
/
Histidina
Idioma:
En
Revista:
Protein Expr Purif
Asunto de la revista:
BIOLOGIA MOLECULAR
Año:
2009
Tipo del documento:
Article
País de afiliación:
Estados Unidos