Expression of cone photoreceptor cGMP-phosphodiesterase α'subunit in Chinese hamster ovary, 293 human embryonic kidney, and Y79retinoblastoma cells

[摘要] Purpose: A functional protein is required for structure/functionanalysis of cone photoreceptor cGMP-phosphodiesterase α' subunit(PDEα'). The purpose of this study was to express enzymaticallyactive PDEα'.Methods: Three expression vectors were constructed for transient andstable expression of PDEα': pC57 (transient) was obtained bysubcloning bovine PDEα' cDNA into the pCIS2 expression vector;pNC57 (stable) was constructed by inserting the neo gene controlledby the mouse phosphoglycerate kinase-1 gene promoter into the pC57vector; and pFC57 (transient) was generated by fusing the sequenceencoding the FLAG peptide to the 5' end of the coding region ofPDEα' cDNA. The recombinant plasmid DNAs were introduced intoHEK293, CHO, or Y79 retinoblastoma cells using the calciumphosphate-mediated transfection procedure or lipofectamin. Northern andwestern blot hybridizations were used for RNA and protein analysis,respectively.Results: Northern blots of both HEK293- and CHO-transfected cellsshowed strong expression of a 3 kb transcript corresponding toPDEα'. cGMP-PDE activity measured in homogenates of transiently andstably transfected cells ranged between 1.5 and 2.2 nmol cGMPhydrolyzed/min x mg total protein, a level of PDE activity slightlygreater than that previously reported for the individualrod-photoreceptor PDE subunits transiently-expressed in HEK293 cells.Western blots of these cell homogenates showed a low level of expressedPDEα'. Transfection of Y79 retinoblastoma cells, that have beenshown to express rod and cone PDEs endogenously, with the constructcontaining cone PDEα' cDNA fused to the FLAG peptide resulted in aprotein with no enzymatic activity.Conclusions: Our results demonstrate that both HEK293 and CHO cellsare capable of expressing functionally active cone PDEα'. Highlevel of mRNA transcription and relatively low protein synthesisefficiency indicates the presence of a post-transcriptional controlmechanism regulating overall expression of PDEα' in HEK293 and CHOcells.

[学科分类] 生物化学/生物物理