1. Implementation of semi-automated cloning and prokaryotic expression screening : the impact of SPINE
- Author
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Alzari, Pedro M., 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, Pär, Perrakis, A., Quevillon-Cheruel, S., Tarandeau, F., van Tilbeurgh, H., Unger, T., Luna-Vargas, M. P. A., Velarde, M., Willmanns, M., Owens, Raymond J., Alzari, Pedro M., 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, Pär, Perrakis, A., Quevillon-Cheruel, S., Tarandeau, F., van Tilbeurgh, H., Unger, T., Luna-Vargas, M. P. A., Velarde, M., Willmanns, M., and Owens, Raymond J.
- Abstract
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.
- Published
- 2006
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