Molecular Cloning, Expression And Purification Of Human Photoreceptor CGMP Phosphodiesterase(PDE6) γ Subunit

Second messenger cAMP and cGMP exert principal regulations in various signal transduction pathways and cellular metabolisms.Phosphodiesterase(PDE) is the key linker in signal transduction pathways of cAMP and cGMP,and has a decisive contribution to adjust cAMP and cGMP concentration in cell.Cyclic GMP photoreceptor phosphodiesterase(PDE6) in rod photoreceptors,which takes cGMP as the special substrate and exists in the retina rod cells,is the central effecter enzyme in the vertebrate visual phototransduction cascade of photoreceptor cells. PDE6 is a multisubunits complexes composed with catalytic and inhibitory subunits,of which theγsubunit serves as a regulatory subunit and can inhibit its activity while binding ontoαandβsubunits.In order to get insight into the relationship between the structure and function of PDE6γ,subunit,the recombinant expression system was constructed and elucidated in this study.Polymerase chain reaction(PCR) was used for amplifying the gene encoding human rod PDE6γ.The recombinant expression plasmid pET22b-PDE6γwas constructed by ligating the PCR product into the Nde I and Xho I restriction enzyme sites of vector pET22b.The positive recombinant,verified by restriction endonuclease digestion and DNA sequencing,was transformed into E.coli BL21(DE3) stain for the production of recombinant protein.Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE),denatured gel electrophoresis with urea,matrix-assisted laser desorption ionization-time of flight mass spectrometry(MALDI-TOF-MS) were used for the evaluating and optimizing the expression and purification of recombinant protein.Protein with purity above 90%was achieved by the low temperature induction at 16℃and one-step purification with nickel affinity chromatography.The results obtained in this study laid valuable foundation for the further structural evaluation of human rod PDE6.