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 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.
Alzari
Pedro M
PM
Unité de Biochimie Structurale, Institut Pasteur, 25-28 Rue du Dr Roux, Paris CEDEX 15, France.
Berglund
H
H
Berrow
N S
NS
Blagova
E
E
Busso
D
D
Cambillau
C
C
Campanacci
V
V
Christodoulou
E
E
Eiler
S
S
Fogg
M J
MJ
Folkers
G
G
Geerlof
A
A
Hart
D
D
Haouz
A
A
Herman
M D
MD
Macieira
S
S
Nordlund
P
P
Perrakis
A
A
Quevillon-Cheruel
S
S
Tarandeau
F
F
van Tilbeurgh
H
H
Unger
T
T
Luna-Vargas
M P A
MP
Velarde
M
M
Willmanns
M
M
Owens
Raymond J
RJ
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