Implementation of semi-automated cloning and prokaryotic expression screening: the impact of SPINE.
Acta Crystallogr D Biol Crystallogr
; 62(Pt 10): 1103-13, 2006 Oct.
Article
em En
| MEDLINE
| ID: mdl-17001088
ABSTRACT
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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Base de dados:
MEDLINE
Assunto principal:
Células Procarióticas
/
Clonagem Molecular
/
Proteômica
Tipo de estudo:
Diagnostic_studies
/
Screening_studies
País como assunto:
Europa
Idioma:
En
Ano de publicação:
2006
Tipo de documento:
Article