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Implementation of s...
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Alzari, Pedro M.
(author)
Implementation of semi-automated cloning and prokaryotic expression screening : the impact of SPINE
- Article/chapterEnglish2006
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LIBRIS-ID:oai:DiVA.org:kth-37384
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https://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-37384URI
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https://doi.org/10.1107/S0907444906029775DOI
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http://kipublications.ki.se/Default.aspx?queryparsed=id:1942817URI
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Language:English
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Summary in:English
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Subject category:ref swepub-contenttype
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Subject category:art swepub-publicationtype
Notes
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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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Berglund, H.
(author)
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Berrow, N. S.
(author)
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Blagova, E.
(author)
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Busso, D.
(author)
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Cambillau, C.
(author)
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Campanacci, V.
(author)
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Christodoulou, E.
(author)
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Eiler, S.
(author)
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Fogg, M. J.
(author)
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Folkers, G.
(author)
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Geerlof, A.
(author)
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Hart, D.
(author)
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Haouz, A.
(author)
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Herman, M. D.
(author)
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Macieira, S.
(author)
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Nordlund, PärKarolinska Institutet
(author)
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Perrakis, A.
(author)
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Quevillon-Cheruel, S.
(author)
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Tarandeau, F.
(author)
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van Tilbeurgh, H.
(author)
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Unger, T.
(author)
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Luna-Vargas, M. P. A.
(author)
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Velarde, M.
(author)
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Willmanns, M.
(author)
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Owens, Raymond J.
(author)
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Karolinska Institutet
(creator_code:org_t)
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In:Acta Crystallographica Section D62, s. 1103-11130907-44491399-0047
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Alzari, Pedro M.
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Berglund, H.
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Berrow, N. S.
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Blagova, E.
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Busso, D.
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Cambillau, C.
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show more...
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Campanacci, V.
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Christodoulou, E ...
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Eiler, S.
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Fogg, M. J.
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Folkers, G.
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Geerlof, A.
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Hart, D.
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Haouz, A.
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Herman, M. D.
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Macieira, S.
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Nordlund, Pär
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Perrakis, A.
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Quevillon-Cherue ...
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Tarandeau, F.
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van Tilbeurgh, H ...
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Unger, T.
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Luna-Vargas, M. ...
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Velarde, M.
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Willmanns, M.
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Owens, Raymond J ...
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- NATURAL SCIENCES
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NATURAL SCIENCES
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and Biological Scien ...
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Acta Crystallogr ...
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Royal Institute of Technology
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Karolinska Institutet