Implementation of semi-automated cloning and prokaryotic expression screening: the impact of SPINE
2006 (English)In: Acta Crystallographica Section D: Biological Crystallography, ISSN 0907-4449, E-ISSN 1399-0047, Vol. 62, 1103-1113 p.Article in journal (Refereed) Published
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.
Place, publisher, year, edition, pages
2006. Vol. 62, 1103-1113 p.
IdentifiersURN: urn:nbn:se:kth:diva-37384DOI: 10.1107/S0907444906029775ISI: 000240618000001PubMedID: 17001088ScopusID: 2-s2.0-33749519973OAI: oai:DiVA.org:kth-37384DiVA: diva2:433573