Аңдатпа
Triple-negative breast cancer (TNBC) is an aggressive form of breast cancer and very few therapeutic options are currently available for its treatment. Interestingly, G-protein coupled receptor 161 (GPR161) is expressed in TNBC cells and can activate the mammalian target of the rapamycin complex 1 signaling pathway. GPR161 and Ras GTPase-activating-like protein, a protein involved in intracellular signaling, proliferation, and cellular adhesion, have been shown to genetically interact in human breast cancer cells. Targeting of GPR161 by monoclonal antibodies may therefore be a strategy to develop diagnostics and therapeutics for TNBC. Thus, to obtain such monoclonal antibodies, we synthesized the GPR161 gene de novo, cloned it into the pET32 expression plasmid, and used the recombinant plasmid to transform the competent BL21 (DE3) strain of Escherichia coli. The recombinant GPR161 gene was designed to contain an N-terminal thioredoxin tag, a thrombin site, the GPR161 sequence, and a C-terminalhexa-histidine tag to facilitate purification by metal-affinity chromatography. Following purification of the recombinant GPR161 (rGPR161) protein using a HisTrap column, we characterized the protein by Western blotting and mass spectrometry. The rGPR161 protein had a molecular mass of ~49 kDa and its identity as rGPR161 was confirmed by mass spectrometry data using the SwissProt database and the Mascot program. Future studies will involve the development of monoclonal antibodies using rGPR161 as the immunogen.