EpCAM and Trop2 are paralogous type I transmembrane glycoproteins expressed at low levels in epithelial and embryonic cells. They are often overexpressed in various carcinomas, making them of considerable interest as potential prognostic markers and therapeutic targets. The two proteins are highly similar in many aspects. They share a high degree of amino acid sequence identity and possess identical domain folding. They play important roles in cell adhesion, signaling, and proliferation, but the exact mechanisms of their function remain poorly understood. Both proteins are found on the cell surface in the form of homodimers. Since EpCAM and Trop2 are co-expressed on the surface of certain cell types, it might be possible that they also form a heterodimer. Molecular dynamics simulations suggest that heterodimer formation would be thermodynamically favourable, with strong interactions between the subunits. A previous attempt to produce the heterodimer in vivo when expression was done in insect cells using a polycistronic baculovirus expressing the ectodomains of EpCAM and Trop2 under different promoters was unsuccessful. Consequently, we altered the experimental approach and attempted to produce the heterodimer by co-infecting Sf9 insect cells. For this purpose, we prepared two monocistronic baculoviruses encoding the Trop2 ectodomain tagged with a Strep-tag II, and the other encoding the EpCAM ectodomain with a polyhistidine tag, both under the control of the polyhedrin promoter. After expression, we attempted to isolate the potential heterodimer by sequential affinity chromatography with nickel affinity chromatography, followed by Strep-Tactin chromatography, which should theoretically result in only the heterodimer in the final eluted fraction. Analysis of the final bound fraction by SDS-PAGE and chemiluminescent western blot detection showed the presence of both ectodomains, present in very low amounts and with a noticeable excess of Trop2. Although our results suggest the formation of the heterodimer, the low yield and lack of appropriate controls prevent us from definitively confirming its existence.
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