A versatile ligation-independent cloning method suitable for high-throughput expression screening applications.
Nucleic Acids Res
; 35(6): e45, 2007.
Article
em En
| MEDLINE
| ID: mdl-17317681
ABSTRACT
This article describes the construction of a set of versatile expression vectors based on the In-Fusion cloning enzyme and their use for high-throughput cloning and expression screening. Modifications to commonly used vectors rendering them compatible with In-Fusion has produced a ligation-independent cloning system that is (1) insert sequence independent (2) capable of cloning large PCR fragments (3) efficient over a wide (20-fold) insert concentration range and (4) applicable to expression in multiple hosts. The system enables the precise engineering of (His(6)-) tagged constructs with no undesirable vector or restriction-site-derived amino acids added to the expressed protein. The use of a multiple host-enabled vector allows rapid screening in both E. coli and eukaryotic hosts (HEK293T cells and insect cell hosts, e.g. Sf9 cells). These high-throughput screening activities have prompted the development and validation of automated protocols for transfection of mammalian cells and Ni-NTA protein purification.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Proteínas Recombinantes de Fusão
/
Clonagem Molecular
Tipo de estudo:
Diagnostic_studies
/
Screening_studies
Limite:
Animals
/
Humans
Idioma:
En
Ano de publicação:
2007
Tipo de documento:
Article