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Nucleic Acids Res. 2007;35(6):e45. Epub 2007 Feb 22.

A versatile ligation-independent cloning method suitable for high-throughput expression screening applications.

Author information

1
The Oxford Protein Production Facility, Henry Wellcome Building for Genomic Medicine, University of Oxford, Oxford, UK.

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.

PMID:
17317681
PMCID:
PMC1874605
DOI:
10.1093/nar/gkm047
[Indexed for MEDLINE]
Free PMC Article

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