Structural characterization of recombinant human gastrokine-1: biochemical properties of the purified protein
Mallardo, Paolo (2011) Structural characterization of recombinant human gastrokine-1: biochemical properties of the purified protein. [Tesi di dottorato] (Inedito)
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Gastric cancer (GC) is one of the most leading cause of cancer death. The high mortality is due to late diagnosis, although the gastric infection by Helicobacter pylori represents one of the most significant risk factors for this type of cancer. Gastrokine-1 (GKN1) is expressed in normal gastric tissue, while it is scarcely present in samples from patients infected by H. pylori, and completely absent in GC tissues and cell lines. Therefore, GKN1 could be a potential marker for GC diagnosis and a possible tumor suppressor. In order to get more insights into the molecular mechanisms on the pathology of GC, this study was directed to better understand the structural features and the biological activity of this protein. In details, this study was directed to: 1) synthesis of recombinant mature GKN1 by cloning, expression and purification; 2) characterization of structural and biochemical properties of recombinant GKN1 by circular dichroism, fluorescence spectroscopy, and limited proteolysis; 3) evaluation of the effect of recombinant GKN1 on gastric cancer cell lines growth; 4) construction of a 3D structural model of the protein. The first protocol for biosynthesis and purification of native human GKN1 in the homologous expression system of Pichia pastoris was settled. The use of biochemical analytical methods such as limited proteolysis led to the identification of exposed amino acid residues on the protein surface. The resistance of GKN1 to the action of proteolytic enzymes was somehow explanatory of its stability in the harsh stomach environment. Spectroscopic studies (fluorescence and circular dichroism) showed that the protein is endowed by a non-proper globular structure due probably to the presence of two domains. These domains showed a different behaviour toward chemical and physical denaturant. The results well correlate with predicted GKN1 secondary structure and 3D structure model. Finally, the recombinant protein showed anti-proliferative properties on gastric cancer cell lines. Our findings contribute to a preliminary clarification of the role of GKN1 in the pathogenesis of gastric cancer.
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