Expression, purification and crystallization attempts of a functional domain of human transforming growth factor beta receptor II - structural characterization of bile acid-binding proteins (Doctoral Thesis)

The aim of this thesis work was to determine the three-dimensional structure of the cytoplasmatic domain of the transforming growth factor β receptor II, and of zebrafish and human ileal bile acid-binding proteins using the X-ray diffraction technique. The expression of TGFBR2 was attempted using different expression systems (E. coli, Pichia pastoris, insect cells transfected with recombinant baculovirus and Nicotiana benthamiana) but only a low amount of soluble recombinant protein was obtained. A refolding method was used to obtain the soluble protein from inclusion bodies and using the refolded protein two microcrystals were found in the preliminary crystallization trials, but they were not suitable for X-ray diffraction experiments. The optimization trials did not produce better samples than microcrystals. Human ileal bile acid-binding protein (HiBABP) and zebrafish ileal bile acid-binding protein (ZiBABP) were expressed in E. coli and purified by immobilized metal ion affinity chromatography, using the hexa-histidine tag added to the C-terminus of the proteins. The three dimensional structure of ZiBABP was determinated both in its apo-form and bound to cholic acid by the molecular replacement method. The resolution was 1.6 Ǻ for the apo-form and 2.2 Ǻ for the two different crystal forms of the complex with cholate. This is the first crystallographic structure of an Ileal bile acid-binding protein. In the case of HiBABP, crystals were obtained in two different crystallization conditions, but their size and quality did not allow to proceed with the structure determination. Optimization experiments of the crystallization conditions are currently being carried out to improve the quality of those crystals. A recent study has shown the presence of a new variant of HiBABP called Human Ileal bile acid-binding protein long (HiBABP-L) and therefore another goal of this thesis work was to determine the three-dimensional structure of this protein. HiBABP-L was expressed in E. coli and attempts to purify the protein of interest are still in progress.