Production of singleton proteins (expression, purification and crystallization) for structural genomics

Abstract

The main objective of this thesis was to prepare samples (soluble proteins and crystals) of selected ORFan proteins for structure determination by X-ray crystallography. ORFan proteins are the proteins having no homologous sequence with any other proteins in other known organisms. Hence for ORFan proteins, structure prediction is not reliable and the only possible way to identify the protein family, fold is the determination of their three-dimensional structures. In this work, a select set of ORFan proteins from Pyrococcus furiosus (ORF PF0772), Aeropyrum pernix K1 (ORFs AP0305, AP0436, AP0371) and Clostridium thermocellum and (ORF Cthe_3042) have been expressed, purified and crystallized. Several crystallization techniques were employed to improve the quality of the diffraction of the crystals. Both seleneo-methionyl (Se-Met) incorporation and heavy atom soaking were used to provide initial phase information. Diffraction quality crystals were obtained for AP0305, AP0436, AP0371 and Cthe_3042. The structure of Cthe_3042 has been determined to 2.1Å resolution using Se-Met labeled protein.