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

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Alzari, Pedro Maria | Berglund, H. | Berrow, N. S. | Blagova, E. | Busso, D. | Cambillau, C. | Campanacci, V. | Christodoulou, E. | Eiler, S. | Fogg, M. J. | Folkers, G. | Geerlof, A. | Hart, D. | Haouz, A. | Herman, M. D. | Macieira, S. | Nordlund, P. | Perrakis, A. | Quevillon-Cheruel, S. | Tarandeau, F. | van Tilbeurgh, H. | Unger, T. | Luna-Vargas, M. P. A. | Velarde, M. | Willmanns, M. | Owens, Raymond J

Edité par CCSD ; International Union of Crystallography -

International audience. 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.

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