title: Cloning, production, and purification of proteins for a medium-scale structural genomics project.
abstract: The South-Paris Yeast Structural Genomics Pilot Project (http://www.genomics.eu.org) aims at systematically expressing, purifying, and determining the three-dimensional structures of Saccharomyces cerevisiae proteins. We have already cloned 240 yeast open reading frames in the Escherichia coli pET system. Eighty-two percent of the targets can be expressed in E. coli, and 61% yield soluble protein. We have currently purified 58 proteins. Twelve X-ray structures have been solved, six are in progress, and six other proteins gave crystals. In this chapter, we present the general experimental flowchart applied for this project. One of the main difficulties encountered in this pilot project was the low solubility of a great number of target proteins. We have developed parallel strategies to recover these proteins from inclusion bodies, including refolding, coexpression with chaperones, and an in vitro expression system. A limited proteolysis protocol, developed to localize flexible regions in proteins that could hinder crystallization, is also described.
authors.affiliation: Institut de Biochimie et Biophysique Moléculaire et Cellulaire, Université de Paris-Sud, Orsay, France.
authors.forename: Sophie
authors.initials: S
authors.lastname: Quevillon-Cheruel
authors.forename: Bruno
authors.initials: B
authors.lastname: Collinet
authors.forename: Lionel
authors.initials: L
authors.lastname: Trésaugues
authors.forename: Philippe
authors.initials: P
authors.lastname: Minard
authors.forename: Gilles
authors.initials: G
authors.lastname: Henckes
authors.forename: Robert
authors.initials: R
authors.lastname: Aufrère
authors.forename: Karine
authors.initials: K
authors.lastname: Blondeau
authors.forename: Cong-Zhao
authors.initials: CZ
authors.lastname: Zhou
authors.forename: Dominique
authors.initials: D
authors.lastname: Liger
authors.forename: Nabila
authors.initials: N
authors.lastname: Bettache
authors.forename: Anne
authors.initials: A
authors.lastname: Poupon
authors.forename: Ilham
authors.initials: I
authors.lastname: Aboulfath
authors.forename: Nicolas
authors.initials: N
authors.lastname: Leulliot
authors.forename: Joël
authors.initials: J
authors.lastname: Janin
authors.forename: Herman
authors.initials: H
authors.lastname: van Tilbeurgh
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D019295:Computational Biology; D003460:Crystallization; D023281:Genomics; D010447:Peptide Hydrolases / Q000378:metabolism; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D011506:Proteins; D011994:Recombinant Proteins; D010447:Peptide Hydrolases