Implementation of semi-automated cloning and prokaryotic expression screening: the impact of SPINE.
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...
| Main Authors: | , , , , , , , , , , , , , , , , , , , , , , , , |
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| Format: | Journal article |
| Language: | English |
| Published: |
2006
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| _version_ | 1826262041053102080 |
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| author | Alzari, P Berglund, H Berrow, N Blagova, E Busso, D Cambillau, C Campanacci, V Christodoulou, E Eiler, S Fogg, M Folkers, G Geerlof, A Hart, D Haouz, A Herman, MD Macieira, S Nordlund, P Perrakis, A Quevillon-Cheruel, S Tarandeau, F van Tilbeurgh, H Unger, T Luna-Vargas, M Velarde, M Willmanns, M |
| author_facet | Alzari, P Berglund, H Berrow, N Blagova, E Busso, D Cambillau, C Campanacci, V Christodoulou, E Eiler, S Fogg, M Folkers, G Geerlof, A Hart, D Haouz, A Herman, MD Macieira, S Nordlund, P Perrakis, A Quevillon-Cheruel, S Tarandeau, F van Tilbeurgh, H Unger, T Luna-Vargas, M Velarde, M Willmanns, M |
| author_sort | Alzari, P |
| collection | OXFORD |
| description | 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. |
| first_indexed | 2024-03-06T19:30:05Z |
| format | Journal article |
| id | oxford-uuid:1d27908d-c35a-4aeb-ba3a-848a78804c5d |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-06T19:30:05Z |
| publishDate | 2006 |
| record_format | dspace |
| spelling | oxford-uuid:1d27908d-c35a-4aeb-ba3a-848a78804c5d2022-03-26T11:09:21ZImplementation of semi-automated cloning and prokaryotic expression screening: the impact of SPINE.Journal articlehttp://purl.org/coar/resource_type/c_dcae04bcuuid:1d27908d-c35a-4aeb-ba3a-848a78804c5dEnglishSymplectic Elements at Oxford2006Alzari, PBerglund, HBerrow, NBlagova, EBusso, DCambillau, CCampanacci, VChristodoulou, EEiler, SFogg, MFolkers, GGeerlof, AHart, DHaouz, AHerman, MDMacieira, SNordlund, PPerrakis, AQuevillon-Cheruel, STarandeau, Fvan Tilbeurgh, HUnger, TLuna-Vargas, MVelarde, MWillmanns, MThe 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. |
| spellingShingle | Alzari, P Berglund, H Berrow, N Blagova, E Busso, D Cambillau, C Campanacci, V Christodoulou, E Eiler, S Fogg, M Folkers, G Geerlof, A Hart, D Haouz, A Herman, MD Macieira, S Nordlund, P Perrakis, A Quevillon-Cheruel, S Tarandeau, F van Tilbeurgh, H Unger, T Luna-Vargas, M Velarde, M Willmanns, M Implementation of semi-automated cloning and prokaryotic expression screening: the impact of SPINE. |
| title | Implementation of semi-automated cloning and prokaryotic expression screening: the impact of SPINE. |
| title_full | Implementation of semi-automated cloning and prokaryotic expression screening: the impact of SPINE. |
| title_fullStr | Implementation of semi-automated cloning and prokaryotic expression screening: the impact of SPINE. |
| title_full_unstemmed | Implementation of semi-automated cloning and prokaryotic expression screening: the impact of SPINE. |
| title_short | Implementation of semi-automated cloning and prokaryotic expression screening: the impact of SPINE. |
| title_sort | implementation of semi automated cloning and prokaryotic expression screening the impact of spine |
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