Gastrokine-1 (GKN1), a protein expressed in normal gastric tissue, but absent in gastric cancer tissues and derived cell lines, has recently emerged as a potential biomarker for gastric cancer. To better establish the molecular properties of GKN1, the first protocol for the production of mature human GKN1 in the expression system of Pichia pastoris was settled. The recombinant protein showed anti-proliferative properties specifically on gastric cancer cell lines thus indicating that it was properly folded. Characterization of structural and biochemical properties of recombinant GKN1 was achieved by limited proteolysis analysis, circular dichroism and fluorescence spectroscopy. The analysis of GKN1 primary structure coupled to proteolytic experiments highlighted that GKN1 was essentially resistant to proteolytic enzymes and showed the presence of at least a disulphide bond between Cys61 and one of the other three Cys (Cys122, Cys145 and Cys159) of the molecule. The secondary structure analysis revealed a prevailing β-structure. Spectroscopic and calorimetric investigations on GKN1 thermal denaturation pointed out its high thermal stability and suggested a more complex than a two-state unfolding process. The resulting protein was endowed with a globular structure characterized by domains showing different stabilities toward chemical and physical denaturants. These results are in agreement with the prediction of GKN1 secondary structure and a three-dimensional structure model. Our findings provide the basis for the development of new pharmaceutical compounds of potential use for gastric cancer therapy.

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