Expression optimization and purification of a recombinant fragment of the GPR161 receptor expressed in Escherichia coli

Abstract

The cell-surface GPR161 molecule is an essential regulator of the proliferation and migration of cancer cells. This molecule serves as a receptor for breast cancer cells and is defined as a prognostic biomarker for triple-negative breast cancer. Despite Escherichia coli being an inexpensive and reliable producer or recombinant proteins, obtaining sufficient amounts of soluble GPR161 for structural studies or producing monoclonal antibodies are hampered by difficulties stemming from a generally poor expression of eukaryotic membrane proteins in bacteria and improper protein folding that render the protein insoluble. In this study a soluble fusion protein comprising the extracellular domain of GPR161 was produced in quantities sufficient for biochemical and structural studies using bacterial expression. Recombinant protein expression and purification protocols were optimized including the selection of solubility enhancing fusion-partner proteins, modifying genetic designs and culture conditions. The E. coli strains BL21(DE3) and ArcticExpress(DE3) were used. As a result of the expression construct optimization, a fragment of the GPR161 protein and thioredoxin were selected as partner proteins to express a soluble fusion protein. Plasmids pET32/Trx+4-5GPR161 and pET28/4-5GPR161+Trx were assembled and transformed into E. coli strains to develop producer strains BL21/pET32/4-5GPR161 and BL21/pET28/4-5GPR161+Trx. The optimized culture and recombinant protein purification protocols allow obtaining the fusion protein at concentrations up to 200 μg/mL. The results of this study are helpful for constructing expression systems recombinant GPCR molecules and other membrane proteins.

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