فهرست مطالب فاطمه نعمتی نیکو

Background and Calprotectin is a participant factor in innate immune system which, with its subunits, interferes in inflammatory and tumorigenesis processes. This study performed in order to evaluate thermal stability of native and modified recombinant calprotectin subunits in presence of Calcium ligand. Methods and materials: Recombinant subunitsin two native and modified forms, S100A8 & S100A9, were denatured by fluorescence spectroscopy in the temperature range of 20-95˚c after incubation with Calcium. Using Excel program, Gibbs free energy changes and Tm were calculated with fluorescence intensity of native and denatured forms and the stability of different forms of protein was indicated. The chart of fluorescence intensity against temperature changes in three forms, native, DEP modified and Calcium incubated ones. Also the calculateed Gibbs free energy showed increasing stability of both DEP modified and Calcium incubated protein in comparison with native form. Stability or instability of protein affects its performance and it can be useful or harmful. According to dual properties of calprotectin and its subunits in cancer process, structure and stability changes of this protein can be affect its performance in cancer process. Therefore, study of calprotectin can be useful for inhibition of cancer by a natural resource.

S100A8 as a subunit of calprotectin heterodimer plays a role in inflammatory processes and cancer. The aim of this study was to express recombinant S100A8 and to evaluate Ca effect on its tertiary structure. This experimental study was performed in Qazvin University of medical sciences, 2013. Recombinant S100A8 subunit was expressed in pET15b, E. coli BL21 (DE3) system as a his-tagged protein. The protein purification process was accomplished under both native and denaturing conditions using Ni-NTA column and different concentrations of imidazole. The expression and homogeneity of recombinant protein was analyzed using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The tertiary structure of S100A8 was studied in the absence and presence of Ca using fluorescence spectrometry. The highest expression of S100A8 with apparent molecular weight of 8 kDa was found in denaturing conditions. In the purification process, the most purified S100A8 was seen with a gradient of 150 and 200 mM imidazole. The maximum fluorescence emission of S100A8 was observed at 330 nm in the presence and absence of calcium. Emission intensity was decreased in the presence of different concentrations of calcium. Changes in tertiary structure of S100A8 subunit in the presence of calcium may affect the protein function and may contribute to understand the role of this protein in cancer and inflammatory processes.