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© Institut Pasteur
Cells infected for 24 hrs with C. Trachomatis. The cell nuclei are labelled in blue, the bacteria appear yellow, within the inclusion lumen. A bacterial protein secreted out the inclusion into the host cytoplasm id labelled in red.
Publication : Acta crystallographica. Section D, Biological crystallography

Implementation of semi-automated cloning and prokaryotic expression screening: the impact of SPINE

Team: Structural Microbiology
Scientific Fields
Diseases
Organisms
Applications
Technique

Published in Acta crystallographica. Section D, Biological crystallography - 19 Sep 2006

Alzari PM, Berglund H, Berrow NS, Blagova E, Busso D, Cambillau C, Campanacci V, Christodoulou E, Eiler S, Fogg MJ, Folkers G, Geerlof A, Hart D, Haouz A, Herman MD, Macieira S, Nordlund P, Perrakis A, Quevillon-Cheruel S, Tarandeau F, van Tilbeurgh H, Unger T, Luna-Vargas MP, Velarde M, Willmanns M, Owens RJ

Link to Pubmed [PMID] – 17001088

Acta Crystallogr. D Biol. Crystallogr. 2006 Oct;62(Pt 10):1103-13

The implementation of high-throughput (HTP) cloning and expression screening in Escherichia coli by 14 laboratories in the Structural Proteomics In Europe (SPINE) consortium is described. Cloning efficiencies of greater than 80% have been achieved for the three non-ligation-based cloning techniques used, namely Gateway, ligation-indendent cloning of PCR products (LIC-PCR) and In-Fusion, with LIC-PCR emerging as the most cost-effective. On average, two constructs have been made for each of the approximately 1700 protein targets selected by SPINE for protein production. Overall, HTP expression screening in E. coli has yielded 32% soluble constructs, with at least one for 70% of the targets. In addition to the implementation of HTP cloning and expression screening, the development of two novel technologies is described, namely library-based screening for soluble constructs and parallel small-scale high-density fermentation.