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Sökning: WFRF:(Alzari P.)

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1.
  • Fogg, M. J., et al. (författare)
  • Application of the use of high-throughput technologies to the determination of protein structures of bacterial and viral pathogens
  • 2006
  • Ingår i: Acta Crystallographica Section D. - 0907-4449 .- 1399-0047. ; 62:10, s. 1196-1207
  • Tidskriftsartikel (refereegranskat)abstract
    • The Structural Proteomics In Europe (SPINE) programme is aimed at the development and implementation of high-throughput technologies for the efficient structure determination of proteins of biomedical importance, such as those of bacterial and viral pathogens linked to human health. Despite the challenging nature of some of these targets, 175 novel pathogen protein structures (approximately 220 including complexes) have been determined to date. Here the impact of several technologies on the structural determination of proteins from human pathogens is illustrated with selected examples, including the parallel expression of multiple constructs, the use of standardized refolding protocols and optimized crystallization screens.
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3.
  • Alzari, Pedro M., et al. (författare)
  • Implementation of semi-automated cloning and prokaryotic expression screening : the impact of SPINE
  • 2006
  • Ingår i: Acta Crystallographica Section D. - 0907-4449 .- 1399-0047. ; 62, s. 1103-1113
  • Tidskriftsartikel (refereegranskat)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.
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