Chapter 2: Cloning, Production, and Purification of Proteins for a Medium-Scale Structural Genomics Project
Sophie Quevillon-Cheruel, Bruno Collinet, Lionel Trésaugues, Philippe Minard, Gilles Henckes, Robert Aufrère, Karine Blondeau, Cong-Zhao Zhou; Dominique Liger, Nabila Bettache, Anne Poupon, Ilham Aboulfath, Nicolas Leulliot, Joël Janin & Herman van Tilbeurgh
he 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.