I’m expressing the N- and P-domains of a membrane protein (only the cytosolic regions) using a FLAG-tagged construct in the pET28b vector, transformed into BL21(DE3) cells. The cultures were grown overnight at 20 °C and induced with various IPTG concentrations.

In the attached SDS-PAGE gel, I pelleted 1 mL culture, boiled the pellets directly in SDS at 95 °C, and loaded them. There’s a faint band at ~55 kDa (the expected size), but in the induced samples I see material stuck at the top of the gel wells, which looks like some form of aggregation.

To try to improve solubility, I followed a lysis protocol using lysozyme, Triton X-100, and DNase, without sonication (I assumed this would be sufficient for 1 mL of cells). After centrifugation, I treated the insoluble pellet with 8 M urea, centrifuged again, and boiled the supernatant in SDS at 70 °C, but the gel came out completely clear, with no band at all.

Has anyone seen this type of aggregation at the top of wells before? Could it indicate inclusion body formation, even though the construct contains only cytosolic domains?

I’d appreciate any advice on interpreting these results or improving solubility.