Parallel Cloning, Expression, Purification, Crystallization Of Human Proteins For Structural Genomics

Following the completion of the human genome sequence, much attention is now shifting towards the functions of the gene products. The highput through crystallization and determination of unknown function protein was a main approaches to explain their function.100 human genes were selected as test targets for parallel cloning, expression, purification and crystallization. Proteins from these genes were selected to have a molecular weight of between 20 and 50 k Da, not to have a high percentage of hydrophobic residues (i.e. more likely to be soluble) and to have no known crystal structures and were not known to be subunits of heterocomplexes. To date, 100 expression clones have been constructed with the GatewayTM cloning system. Of these, 79 clones were expressed as recombinant proteins in Escherichia coli strain BL21(DE3), and 9 clones were expressed as recombinant proteins in Escherichia coli strain Rosetta(DE3), of which 21 were soluble and 12 have been puried to homogeneity. Crystallization conditions were screened for the purified proteins in 48-well plates by the sitting-drop. After further refinement with the same device or by the hanging-drop method, The crystal of COQ3 were grown. The results provide insights into the structure and function of those protein.