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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.

Original publication

DOI

10.1093/nar/gkm047

Type

Journal article

Journal

Nucleic Acids Res

Publication Date

2007

Volume

35

Keywords

Animals, Bacterial Proteins, Cell Line, Cloning, Molecular, Escherichia coli, Genes, Viral, Genetic Vectors, Humans, Neisseria, Nerve Tissue Proteins, Recombinant Fusion Proteins, Transcription Factors