Implementation of semi-automated cloning and prokaryotic expression screening: the impact of SPINE

被引：58
作者：
Alzari, Pedro M.
Berglund, H.
Berrow, N. S.
Blagova, E.
Busso, D.
Cambillau, C.
Campanacci, V.
Christodoulou, E.
Eiler, S.
Fogg, M. J.
Folkers, G.
Geerlof, A.
Hart, D.
Haouz, A.
Herman, M. D.
Macieira, S.
Nordlund, P.
Perrakis, A.
Quevillon-Cheruel, S.
Tarandeau, F.
van Tilbeurgh, H.
Unger, T.
Luna-Vargas, M. P. A.
Velarde, M.
Willmanns, M.
Owens, Raymond J.
机构：
[1] Wellcome Trust Ctr Human Genet, Oxford Prot Prod Facil, Oxford OX3 7BN, England
[2] Wellcome Trust Ctr Human Genet, Div Struct Biol, Oxford OX3 7BN, England
[3] Inst Pasteur, Unite Biochim Struct, F-75724 Paris 15, France
[4] Karolinska Inst, Dept Med Biochem & Biophys, SE-10951 Stockholm, Sweden
[5] Univ York, York Struct Biol Lab, Dept Chem, York YO10 5YW, N Yorkshire, England
[6] Inst Genet & Biol Mol & Cellulaire, F-67404 Illkirch Graffenstaden, France
[7] CNRS Univ Provence Univ Mediterranee, CNRS, Architecture & Fonct Macromol Biol UMR 6098, F-13288 Marseille 09, France
[8] Netherlands Canc Inst, Div Mol Carcinogenesis, NL-1066 CX Amsterdam, Netherlands
[9] Univ Utrecht, Bijvoet Ctr Biomol Res, NL-3584 CH Utrecht, Netherlands
[10] EMBL, Hamburg Outstn, D-22603 Hamburg, Germany
[11] ILL Grenoble, EMBL Grenoble, F-38042 Grenoble 9, France
[12] Max Planck Inst Biochem, Dept Proteom & Signal Transduct, D-82152 Martinsried, Germany
[13] Univ Stockholm, AlbaNova Ctr, Royal Inst Technol, Dept Biotechnol, S-10691 Stockholm, Sweden
[14] Univ Paris Sud, Inst Biochim & Biophys Mol & Cellulaire, UMR8619, F-91405 Orsay, France
[15] Weizmann Inst Sci, Israel Struct Proteom Ctr, Dept Biol Struct, IL-76100 Rehovot, Israel
来源：
ACTA CRYSTALLOGRAPHICA SECTION D-STRUCTURAL BIOLOGY | 2006年 / 62卷
基金：
英国医学研究理事会;
关键词：
D O I：
10.1107/S0907444906029775
中图分类号：
Q5 [生物化学];
学科分类号：
071010 ; 081704 ;
摘要：
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.
引用
收藏
页码：1103 / 1113
页数：11
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