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T. Iwata, N. Sanuki, Y. Mashima, Y. Tanaka; Analysis of Optineurin-RAB8 Protein Interaction Uisng Quartz-Crystal Microbalance (QCM) . Invest. Ophthalmol. Vis. Sci. 2003;44(13):1114.
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Purpose: Recently Sarfarazi et al. (Science 2002) identified a new gene optineurin (OPTN) responsible for primary open-angle glaucoma and normal tension glaucoma. This protein was previously characterized to interact with several proteins including RAB8. To further characterize the effect of mutation to OPTN-RAB8 protein interaction, Quartz-Crystal Microbalance (QCM), a new method to analyze protein-protein interaction was applied. Methods: Human OPTN was amplified by RT-PCR from human kidney total RNA and cloned into N-terminal His-tag bacterial expression vector pTrcHis (Invitrogen). Mutations 458G>A, 691_692insAG, 1944G>A, and SNP 603T>A were introduced with Quick Change Multi Site-Directed Mutagenesis Kit (Stratagene). Bacteria TOP10 was transformed with each OPTN construct and incubated for 6h under 1mM IPTG. OPTN was purified by affinity (HiTrap Chelating HP) and gel filtration (Superdex-75 HR 10/30) chromatography with HPLC AKTA Explorer (Amersham Biosciences). RAB8 was amplified by RT-PCR from brain total RNA and cloned for expression. Purification was performed by the same procedure. Quartz-Crystal Microbalance (AffinixQ, Initium) was used to measure protein-protein interaction of two purified protein in PBS solution containing blocking reagent for AffinixQ. Thirty to three hundred nanograms of purified RAB8 were attached to QCM chip. Various amount of OPTN dissolved in 8ul of 20mM phosphate buffer (pH7.6) was injected into 8ml PBS solution in QCM interaction chamber for interaction measurement. Results: All the constructs with mutation had significant reduction of protein expression compared with wild type. Mutant OPTN 458G>A showed weak binding to the affinity column presumably due to the conformational change in protein structure. OPTN with mutation had significantly weak affinity to purified RAB8 protein. Detail numbers will be presented at the meeting. Conclusions: QCM, a new method to analyze protein-protein interaction was applied to characterize OPTN-RAB8 protein interaction. Mutations and polymorphism reported by Sarfarazi et al. (Science 2002) were introduced into wild type OPTN cDNA. These OPTN constructs were then expressed and purified for one to one interaction with purified RAB8 protein. Interaction with RAB8 was significantly affected by several mutations.
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