مجله دانشکده پزشکی اصفهان (May 2015)
Construction and Characterization of Recombinant HEK Cell Over-Expressing TOSO/FAIM3 and Evaluation of its Expression
Abstract
Background: Integral membrane proteins involve in a variety of valuable cellular processes including signal transduction, transport and cell-to-cell communication. Thus, due to their importance, are targeted to treatment of diseases or disorders. To achieve this goal, it is necessary to obtain adequate membrane proteins. A variety of expression systems have been used to overexpress these proteins but mammalian cells can produce membrane proteins structurally close to natural form of them and are favourable for this purpose. TOSO/FAIM3 (Fas Apoptosis Inhibitory Molecule), highly conserved membrane protein, playing important role in cell survival, immune surveillance, and homeostasis and in this study was overexpressed on HEK-293T membrane. Methods: Eukaryotic expression vector pEZ-M67-TOSO was transformed into Escherichia coli top 10F’ strain to multiply. Then, the plasmid was extracted and digested with restriction enzyme Eco31I to make a linear plasmid. Subsequently, HEK-293T cells were transfected with linear plasmid and treated with hygromycin to select TOSO expressing stable cells. After three weeks, resistant colons were expanded, Chromosomal DNA and total RNA were extracted and the presence of TOSO-cDNA in HEK-293T genome and the expression level of TOSO were evaluated using polymerase chain reaction (PCR) and Real-time PCR methods, respectively. Findings: The integration of TOSO expressing cassette in HEK-293T genome was confirmed. HEK-293 cells expressed 1700 mRNA TOSO molecule/cell which was in the range of protein with abundant expression. Conclusion: Mammalian cells represent membrane proteins in desirable form versus other expression systems. For this reason, we used HEK293T cells to overexpress transmembrane TOSO protein. In this investigation, we successfully constructed the HEK293 cell line with stable TOSO overexpression, which will facilitate further researches to characterize biophysical and biochemical structure and function of TOSO and pharmaceutical biological researches about it.
