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Hongyu Ying, Xiang Shen, Minhua Wang, Beatrice Yue; Mammalian Expression and Biophysical Examination of Human Wild-Type Optineurin Protein. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1610.
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To express optineurin in mammalian cells and attain highly purified optineurin protein for biophysical examinations. Optineurin is a gene linked to normal tension glaucoma and amyotrophic lateral sclerosis.
Tetracycline inducible (Tet-on) neuronal RGC5 cell lines that express Halo tagged wild-type optineurin (OPTNWT-Halo or optineurin-Halo) were created by transfecting RGC5 cells with plasmid vector pTRE-OPTNWT-Halo-IRES-GFP-INS-rtTA-IRES-hyg-pcDNA3.1z. Cells were selected in hygromycin (100 μg/ml)-containing medium and subsequently screened for optineurin expression by Western blotting or based on GFP expression after doxycycline (Dox) induction by fluorescence microscopy. Optineurin-Halo collected from the cell lysate was purified using Promega’s HaloTag based affinity purification technique under native conditions. The purity of the isolated protein was assessed by SDS-PAGE and Western blotting. The secondary structure of purified optineurin was examined by Jasco 710 circular dichroism (CD) spectropolarimeter.
Tet-on inducible RGC5 cell lines were established to express optineurin-Halo upon Dox induction. Following affinity purification, Western blotting with anti-optineurin antibody detected a single band at approximately 74 kDa. This highly purified tag-free optineurin protein was isolated with a yield of 23 µg/500 cm2 (22 cm square dish). A circular dichroism (CD) spectrum indicated that optineurin is folded, containing both α-helical and β-sheet secondary structures.
Human optineurin protein expressed in stable inducible cell lines was purified and isolated. The inducible cell lines would allow production of highly purified protein in a scaled-up and cost effective manner, facilitating biophysical characterization of optineurin including its secondary structure, thermal stability and aggregation.
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