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H. Sakai, B.C. Park, X. Shen, B.Y. J. T. Yue; Transduction of TAT–Fusion Proteins into Human and Bovine Trabecular Meshwork . Invest. Ophthalmol. Vis. Sci. 2005;46(13):3701.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To examine the potential of using the TAT–fusion protein technology to transduce TAT–ß–galactosidase and TAT–myocilin proteins into trabecular meshwork (TM) cells in culture and in tissues. Methods: TAT–ß–galactosidase and TAT–myocilin fusion proteins were expressed in E. coli and purified using Ni–NTA agarose columns. Normal human TM cell cultures, human and bovine TM tissues in organ cultures, and bovine eyes in perfusion organ cultures were incubated or perfused with the TAT fusion proteins. Transduction of TAT–ß–galactosidase into TM cells and tissues was detected by X–gal staining. Transduction of TAT–myocilin was evaluated by immunostaining against myocilin. ß–Galactosidase protein was used as a negative control. Results: Blue staining in the TAT–ß–galactosidase–treated cells or eyes signified ß–galactosidase activity resulting from transduction. The X–gal staining was observed in the cytoplasm of nearly all cells in TM cell cultures in a concentration–dependent manner. TAT–ß–galactosidase was also transduced into cells in various regions of TM tissues in organ cultures. After TAT–myocilin incubation, immunofluorescence using anti–myocilin showed an intensified preinuclear staining compared to controls in TM cell cultures, indicating successful protein transduction. A stronger myocilin staining was likewise noted in both the cells and beams in all TM regions of TAT–myocilin–incubated or perfused eyes. Conclusions: TAT–fusion proteins can be efficiently transduced into TM cells and tissues in a variety of culture systems. The TAT–mediated protein transduction technology may be very useful in functional studies of proteins such as myocilin in the TM.
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