Refolding strategies from inclusion bodies in a structural genomics project.
The South-Paris Yeast Structural Genomics Project aims at systematically expressing, purifying and determining the structure of S. cerevisiae proteins with no detectable homology to proteins of known structure. We brought 250 yeast ORFs to expression in E. coli, but 37% of them form inclusion bodies. This important fraction of proteins that are well expressed but lost for structural studies prompted us to test methodologies to recover these proteins. Three different strategies were explored in parallel on a set of 20 proteins: (1) refolding from solubilized inclusion bodies using an original and fast 96-well plates screening test, (2) co-expression of the targets in E. coli with DnaK-DnaJ-GrpE and GroEL-GroES chaperones, and (3) use of the cell-free expression system. Most of the tested proteins (17/20) could be resolubilized at least by one approach, but the subsequent purification proved to be difficult for most of them.
Trésaugues
Lionel
L
Institut de Biochimie et de Biophysique Moléculaire et Cellulaire (CNRS-UMR 8619), Université Paris-sud, Bât. 430, F-91400 Orsay, France.
Collinet
Bruno
B
Minard
Philippe
P
Henckes
Gilles
G
Aufrère
Robert
R
Blondeau
Karine
K
Liger
Dominique
D
Zhou
Cong-Zhao
CZ
Janin
Joël
J
Van Tilbeurgh
Herman
H
Quevillon-Cheruel
Sophie
S
D016681:Genome, Fungal; D002479:Inclusion Bodies / Q000737:chemistry* / Q000235:genetics; D016366:Open Reading Frames / Q000235:genetics*; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics; D012995:Solubility
D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins