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چکیده
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ADP-ribosylation factor-like protein 13B (Arl13b) is implicated in cilia assembly, particularly in the regulation of ciliary cargo traffic. Sec8 is a subunit of the exocyst complex proteins that regulates the ciliogenesis and proper localization of intracellular membrane vesicles to their sites of fusion with the plasma membrane. SAP102, a member of the membrane-associated guanylate kinase (MAGUK) family of PDZ proteins, has a wider distribution and is more abundant in the cytoplasm. The genes of CrArl13b, Sec8 and Sap102a were amplified by PCR from a cDNA library from C. rheinhardtii then were cloned into MalPET, GST29a and PET15b vectors respectively. All proteins were expressed in Escherichia coli BL21 (DE2) following induction with 100 μM IPTG at 18◦C overnight. Purification was done using Nickel Affinity Gradient Chromatography which was washed with wash buffer [20 mM Tris, pH 8, 100 mM NaCl, 20 mM Imidazole, 10 mM 2-mercaptoethanol and 5% glycerol]. The proteins were eluted with elution buffer [20 mM Tris, pH 8, 100 mM NaCl, 1 M Imidazole, 10 mM 2-mercaptoethanol and 5% glycerol]. Following cleavage CrArl13b-MalpET with TEV (tobacco etch virus), Sec8-GST29a and SAP102a-PET15b with thrombin (overnight), residual MalPET, GST29a and PET15b were removed. The CrArl13b, Sec8 and Sap102a proteins were collected in Flowthrough of a Histrap chromatography that the MalPET, GST29a and PET15b bind to Histrap column. The CrArl13b, Sec8 and Sap102a proteins were purified by size exclusion chromatography (Superdex 200 16/60, equilibrated with gel filtration buffer; 20 mM Tris, pH 8, 100 mM NaCl, 2 mM DTT, 5% glycerol). We mixed Sec8c with Sap102a and CrArl13b separately. The complexes were concentrated and analysis of protein complexes was performed by size exclusion chromatography. The SDS-PAGE results show that Sec8c interacts with Sap102a and CrArl13b separately. These findings suggest that the CrArl13b protein can regulate the SAP102a traffic via Sec8c protein.
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