title: Implementation of semi-automated cloning and prokaryotic expression screening: the impact of SPINE.
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.
authors.affiliation: Unité de Biochimie Structurale, Institut Pasteur, 25-28 Rue du Dr Roux, Paris CEDEX 15, France.
authors.forename: Pedro M
authors.initials: PM
authors.lastname: Alzari
authors.forename: H
authors.initials: H
authors.lastname: Berglund
authors.forename: N S
authors.initials: NS
authors.lastname: Berrow
authors.forename: E
authors.initials: E
authors.lastname: Blagova
authors.forename: D
authors.initials: D
authors.lastname: Busso
authors.forename: C
authors.initials: C
authors.lastname: Cambillau
authors.forename: V
authors.initials: V
authors.lastname: Campanacci
authors.forename: E
authors.initials: E
authors.lastname: Christodoulou
authors.forename: S
authors.initials: S
authors.lastname: Eiler
authors.forename: M J
authors.initials: MJ
authors.lastname: Fogg
authors.forename: G
authors.initials: G
authors.lastname: Folkers
authors.forename: A
authors.initials: A
authors.lastname: Geerlof
authors.forename: D
authors.initials: D
authors.lastname: Hart
authors.forename: A
authors.initials: A
authors.lastname: Haouz
authors.forename: M D
authors.initials: MD
authors.lastname: Herman
authors.forename: S
authors.initials: S
authors.lastname: Macieira
authors.forename: P
authors.initials: P
authors.lastname: Nordlund
authors.forename: A
authors.initials: A
authors.lastname: Perrakis
authors.forename: S
authors.initials: S
authors.lastname: Quevillon-Cheruel
authors.forename: F
authors.initials: F
authors.lastname: Tarandeau
authors.forename: H
authors.initials: H
authors.lastname: van Tilbeurgh
authors.forename: T
authors.initials: T
authors.lastname: Unger
authors.forename: M P A
authors.initials: MP
authors.lastname: Luna-Vargas
authors.forename: M
authors.initials: M
authors.lastname: Velarde
authors.forename: M
authors.initials: M
authors.lastname: Willmanns
authors.forename: Raymond J
authors.initials: RJ
authors.lastname: Owens
mesh_terms: D000595:Amino Acid Sequence; D001331:Automation; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000378:metabolism; D005060:Europe; D005285:Fermentation; D017353:Gene Deletion; D015723:Gene Library; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D011387:Prokaryotic Cells / Q000378:metabolism*; D017510:Protein Folding; D040901:Proteomics / Q000639:trends*; D017421:Sequence Analysis / Q000295:instrumentation / Q000379:methods