半自动克隆与原核表达筛选的实施：SPINE的影响
Implementation of semi-automated cloning and prokaryotic expression screening: the impact of SPINE.
作者信息
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
机构信息
Unité de Biochimie Structurale, Institut Pasteur, 25-28 Rue du Dr Roux, Paris CEDEX 15, France.
出版信息
Acta Crystallogr D Biol Crystallogr. 2006 Oct;62(Pt 10):1103-13. doi: 10.1107/S0907444906029775. Epub 2006 Sep 19.
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.
欧洲结构蛋白质组学（SPINE）联盟的14个实验室描述了在大肠杆菌中进行高通量（HTP）克隆和表达筛选的实施情况。所使用的三种非连接克隆技术，即Gateway、PCR产物的不依赖连接克隆（LIC-PCR）和In-Fusion，克隆效率均超过80%，其中LIC-PCR是最具成本效益的。平均而言，SPINE选择用于蛋白质生产的约1700个蛋白质靶标，每个靶标制备了两个构建体。总体而言，大肠杆菌中的HTP表达筛选产生了32%的可溶性构建体，70%的靶标至少有一个可溶性构建体。除了实施HTP克隆和表达筛选外，还描述了两项新技术的开发，即基于文库的可溶性构建体筛选和平行小规模高密度发酵。
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