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

• Published in: Protein expression and purification 2018-11, Vol.151, p.38-45
• Main Authors: Wang, Li, Fan, Min, Zeng, Chao, Li, Wei, Hu, Qikang, Liu, Wenliang, Huang, Xingchun, Li, Guiyuan, Yu, Fenglei
• Format: Article
• Language: English
• Subjects: Bortezomib - pharmacology; Cell Line; Expression; Humans; Leupeptins - pharmacology; Membrane Proteins - biosynthesis; Membrane Proteins - isolation & purification; Proteasome Endopeptidase Complex - metabolism; Proteasome Inhibitors - pharmacology; Protein degradation; Protein Isoforms - biosynthesis; Protein Isoforms - isolation & purification; Proteolysis; Purification; Recombinant Proteins - biosynthesis; Recombinant Proteins - isolation & purification; Small molecule inhibitor; TMEM8B; Transfection; Tumor Suppressor Proteins - biosynthesis; Tumor Suppressor Proteins - isolation & purification
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2018.06.002

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 × 108 293FT cells. •The PiggyBac system was used to express TMEM8B-a in mammalian cells.•The TMEM8B-a is degraded via the proteasome pathway rather than via calpains or the lysosomal pathway.•SMIs that target the proteasome efficiently induce TMEM8B-a expression.•Twin-Strep-tag and Strep-Tactin XT resin were used to isolate recombinant TMEM8B-a with 90% purity.