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Sökning: WFRF:(Unger E.) > (2005-2009)

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  • 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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