Expression and purification of a rapidly degraded protein， TMEM8B-a， in mammalian cell line.

TMEM8B-a protein is the longer， predominant isoform of the TMEM8B gene product， which is a tumor metastasis suppressor in nasopharyngeal carcinoma (NPC) and lung cancer. TMEM8B-a is rapidly degraded via the proteasome pathway mediated by ezrin in many NPC and lung cancer cell lines， but TMEM8B-a is not ubiquitinated. In this study， we report the recombinant production of full-length modified TMEM8B-a in mammalian cells. We used the PiggyBac transposon system to efficiently generate normal and lung cancer cell lines with stable TMEM8B-a protein expression. 293FT cells were the best host cell line to express TMEM8B-a protein. Then， we treated the stable 293FT cell lines with various small-molecule inhibitors and demonstrated that treatment with MG-132 and bortezomib， which target the proteasome and disrupt its function， could prevent TMEM8B-a degradation and induce protein expression in 293FT cells. Finally， we utilized the combination of Twin-Strep-tag and Strep-Tactin XT resin to successfully purify the TMEM8B-a protein. The final yield was estimated to be approximately 10-20 μg of the purified TMEM8B-a per 3.0 × 10 293FT cells.