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Nucleic Acids Research Advance Access first published online on February 22, 2007
This version published online on March 12, 2007
Nucleic Acids Research, doi:10.1093/nar/gkm047
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© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Methods Online

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

Nick S. Berrow1, David Alderton1, Sarah Sainsbury1, Joanne Nettleship1, Rene Assenberg1, Nahid Rahman1, David I. Stuart1,2 and Raymond J. Owens1,*

1The Oxford Protein Production Facility and 2Division of Structural Biology, Henry Wellcome Building for Genomic Medicine, University of Oxford, Roosevelt Drive, Oxford, OX3 7BN, UK

*To whom correspondence should be addressed. Tel: +44 1865 287748; Fax: +44 1865 287547; Email: ray{at}strubi.ox.ac.uk

Received November 30, 2006. Accepted January 12, 2007.

This article describes the construction of a set of versatile expression vectors based on the In-FusionTM cloning enzyme and their use for high-throughput cloning and expression screening. Modifications to commonly used vectors rendering them compatible with In-FusionTM 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 (His6-) 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.


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