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

Journal article

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.

Authors: Berrow, N; Alderton, D; Sainsbury, S; Nettleship, J; Assenberg, R

• Publication status: Published
• Journal: Nucleic acids research
• Volume: 35
• Issue: 6
• Pages: e45
• Publication date: 2007-01-01
• DOI: 10.1093/nar/gkm047
• EISSN: 1362-4962
• ISSN: 0305-1048
• Language: English
• Keywords: Animals; Cloning, Molecular; Genes, Viral; Escherichia coli; Bacterial Proteins; Cell Line; Humans; Neisseria; Recombinant Fusion Proteins; Transcription Factors; Genetic Vectors; Nerve Tissue Proteins