作者: P. J. Reeves , R. L. Thurmond , H. G. Khorana
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摘要: Stable mammalian cell lines harboring a synthetic bovine opsin gene have been derived from the suspension-adapted HEK293 line. The is under control of immediate-early cytomegalovirus promoter/enhancer in an expression vector that also contains selectable marker (Neo) governed by relatively weak promoter. expressing at high levels are selected growth presence concentrations antibiotic geneticin. Under conditions used for suspension, produced saturated culture more than 2 mg/liter. After reconstitution with 11-cis-retinal, rhodopsin purified to homogeneity single step immunoaffinity column chromatography. Rhodopsin thus prepared (> 90% recovery up 15 microM) indistinguishable rod outer segments following criteria: (i) UV/Vis absorption spectra dark and after photobleaching rate metarhodopsin II decay, (ii) initial rates transducin activation, (iii) phosphorylation kinase. Although migrates slower segment on SDS/polyacrylamide gels, presumably due different N-glycosylation pattern, their mobilities deglycosylation identical. This method has enabled preparation several site-specific mutants comparable amounts.