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CS Ricard; Analysis Of Myocilin Protein Secreted From Cultured Human Optic Nerve Head Astrocytes . Invest. Ophthalmol. Vis. Sci. 2002;43(13):4041.
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Purpose: Mutations in the MYOC gene have been associated with early and late onset primary open angle glaucoma (POAG). The function of myocilin is unknown. Because my laboratory studies the cellular and molecular mechanisms of POAG, I undertook to analyze the protein chemistry of myocilin secreted from normal human optic nerve head astrocytes. This first required development of a purification protocol for obtaining sufficient amounts of myocilin protein for analysis. Methods: Myocilin obtained from spent medium from normal cultured human astrocytes, type 1B, (gift of Dr. M. Rosario Hernandez, Washington University St. Louis) was used to scale-up protein quantity. Metabolically radiolabeled myocilin was used to "spike" the spent medium for the purpose of determining protein recovery. Proteins in the medium were selectively precipitated by different concentrations of polyethylene glycol (PEG) and ammonium sulfate (AmS). Immunoaffinity chromatography was used to purify myocilin, using pAb129 (generous gift of Drs. A. Clark and T. Steely, Alcon Labs, Ft Worth TX). In addition a bacterial system was used to express recombinant myocilin. Myocilin was analyzed by denaturing and non-denaturing gel electrophoresis. Results: No qualitative differences were observed in myocilin that was first concentrated by PEG or by AmS, however PEG precipitation required less manipulation prior to electrophoretic and other analyses. Myocilin was purified from cultured medium by immunoaffinity chromatography with approximately 39% recovery of the radiolabeled material. Conclusion: My results demonstrate that the spent culture medium from ONH astrocytes provides a convenient source of soluble native myocilin in vitro. Purification of myocilin is key to biochemical and biophysical analysis, and for x-ray crystal structure analysis. Such structural studies will provide insight for the function(s) of myocilin in vivo and in POAG.
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