Protein production in Escherichia coli for structural studies by X-ray crystallography.
The arrival of genomic sequences to the database has provided a seemingly unlimited supply of targets for protein structure determination and the possibility of solving the structure of an entire proteome. Based on our experience with the proteomes of Pyrobaculum aerophilum and Mycobacterium tuberculosis, we have developed a simple strategy for the production of proteins for structural studies by X-ray crystallography. Our scheme demonstrates a strong protein target commitment and includes the expression of genes from these organisms in Escherichia coli. These proteins are expressed with affinity tags and purified for characterization and crystallization. We have identified protein solubility and crystallization as the two major bottlenecks in the process toward the determination of protein structures by X-ray diffraction. Strategies to overcome these bottlenecks are discussed.
Goulding
Celia W
CW
UCLA-DOE Center for Genomics and Proteomics, University of California at Los Angeles, Los Angeles, CA 90095-1570, USA.
Perry
L Jeanne
LJ
D003001:Cloning, Molecular / Q000379:methods; D018360:Crystallography, X-Ray / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics*; D014176:Protein Biosynthesis; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry*
D011506:Proteins