Expression and structural studies of insulin-like growth factor binding proteins and the c-terminal domain of perlecan

Insulin-like Growth Factors (IGFs), Insulin-like Growth Factor Binding Proteins (IGFBPs) and Insulin-like Growth Factor Binding Proteins-related Proteins (IGFBPrP). IGF-I and IGF-II are small hormone peptides of 70 and 67 amino acids respectively and are called insulin-like because of their homology with insulin. IGFs promote cellular growth and differentiation, bind IGFBPs in vivo and also two types of receptors (type 1 and type 2 receptor) which mediate their actions. The IGFBPs form a six member family (IGFBP-1 to -6) and are between 216 to 289 amino acids long. They are subdivided into three distinct domains: a conserved amino-terminal cysteine-rich region, a non-conserved midregion, and a conserved carboxy-terminal region. The IGFBPs bind the circulating IGFs thus regulating their concentration, by sequestering them or releasing them in proximity of their target tissues. In addition, IGFBP effects independent of IGFbinding have been demonstrated. Studies with proteolyzed fragments of IGFBPs unable to bind IGFs, have shown their ability to influence cellular adhesion by integrin binding. The IGF system is involved in various processes and many pathological events have been related to its failure. In addition, other proteins have been identified which have the ability to bind IGFs with lower affinity, compared to IGFBPs. These proteins show homology with IGFBPs in their amino-terminal region and are called IGFBP-related proteins. During this thesis, human IGF-II was expressed in Escherichia coli , in the form of inclusion bodies and in its soluble form. The protein from inclusion bodies was solubilized, purified by affinity chromatography (using a 6 histidine tag), and then refolded. The renatured protein was crystallized but with the data obtained from those crystals it was not possible to determine the three dimensional structure. The soluble form was obtained by using a different expression vector and a 6 histidine tag but purification using the tag was not possible due to the low affinity for the chromatography resin. Human IGFBP-3 was also expressed using the methylotrophic yeast Pichia pastoris . The recombinant protein was purified by ion-exchange chromatography and by gel-filtration but the crystallization trials did not yield good crystals. Human IGFBP-rP1 was expressed in Escherichia coli , purified by affinity chromatography and used for preliminary crystallization trials. One crystallization condition gave small crystals which are still not suitable for X-ray diffraction analysis. LG-3 (Laminin G like) domain of the human perlecan Human perlecan is an heparan-sulphate proteoglycan, containing a protein core of 470 kDa with oligosaccharides and heparan sulphate chains attached to it which give a total molecular weight of 800 kDa. Together with collagen and laminin it forms the basement membrane which is a specialized matrix found in epithelium, endothelium and surrounding muscle cells. The carboxy-terminal domain of perlecan (domain V of 85 kDa) is called endorepellin and possesses anti-angiogenic activity The LG-3 domain of human perlecan was expressed in Escherichia coli and purified by affinity chromatography. The purified protein was then crystallized and its molecular structure was determined by X-ray diffraction analysis. α1-Microglobulin. α1-Microglobulin is a lipocalin of 26 kDa and is 183 amino acids long. Human α1-microglobulin is glycosylated in three positions: two of these are of the Nlinked type and the third one is of the O-linked type. α1-Microglobulin is synthesized in the liver as a fusion protein with bikunin from which it is subsequently cleaved and released into plasma. The biological function of α1- microglobulin is related to its ability to negatively modulate the immune system activity thus preventing tissue damage by immunological responses. Mouse α1-microglobulin was expressed as a recombinant protein by using the yeast Pichia pastoris . The protein was purified by ion-exchange chromatography and gel filtration and preliminary crystallization trials were prepared which gave no crystals.