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

✍ Scribed by 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, 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.

• Publisher: International Union of Crystallography
• Year: 2006
• Tongue: English
• Weight: 976 KB
• Volume: 62
• Category: Article
• ISSN: 0907-4449
• DOI: 10.1107/s0907444906029775

✦ Synopsis

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.