RESUMO
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.
Assuntos
Proteínas/metabolismo , Sequência de Aminoácidos , Clonagem Molecular , Biologia Computacional , Cristalização , Genômica , Peptídeo Hidrolases/metabolismo , Proteínas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
In the Pseudomonas bacterial genomes, the PhzF proteins are involved in the production of phenazine derivative antibiotic and antifungal compounds. The PhzF superfamily however also encompasses proteins in all genomes from bacteria to eukaryotes, for which no function has been assigned. We have determined the three dimensional crystal structure at 2.05 A resolution of YHI9, the yeast member of the PhzF family. YHI9 has a fold similar to bacterial diaminopimelate epimerase, revealing a bimodular structure with an internal symmetry. Residue conservation identifies a putative active site at the interface between the two domains. Evolution of this protein by gene duplication, gene fusion and domain swapping from an ancestral gene containing the "hot dog" fold, identifies the protein as a "kinked double hot dog" fold.
Assuntos
Isomerases de Aminoácido/química , Proteínas de Saccharomyces cerevisiae/química , Isomerases de Aminoácido/genética , Isomerases de Aminoácido/isolamento & purificação , Cristalografia por Raios X , Modelos Moleculares , Reação em Cadeia da Polimerase , Conformação Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/isolamento & purificaçãoRESUMO
Crystallization has long been regarded as one of the major bottlenecks in high-throughput structural determination by X-ray crystallography. Structural genomics projects have addressed this issue by using robots to set up automated crystal screens using nanodrop technology. This has moved the bottleneck from obtaining the first crystal hit to obtaining diffraction-quality crystals, as crystal optimization is a notoriously slow process that is difficult to automatize. This article describes the high-throughput optimization strategies used in the Yeast Structural Genomics project, with selected successful examples.
