Expression of mouse rod photoreceptor cGMP phosphodiesterase gamma subunit in bacteria.
We expressed the gamma subunit of mouse rod photoreceptor cGMP phosphodiesterase (PDE) in the bacterial pGFX-2TK expression vector which produces a cleavable 40 kDa fusion protein. The fusion protein can be isolated in a one step procedure by affinity chromatography on glutathione beads. The yield of purified fusion protein is approximately 10 mg from 1 liter of bacterial culture, or about 3 mg of PDE gamma equivalent to the PDE gamma content of approximately 200,000 mouse retinas. Both the fusion protein and the cleaved PDE gamma, to which a short kinase domain remains attached, are biologically active, inhibiting activated PDE in a manner comparable to native PDE gamma. Immobilized PDE gamma binds transducin alpha subunit charged with GTP, PDE alpha and beta subunits, and, unexpectedly, arrestin (S-antigen).
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Department of Biochemistry, Baylor College of Medicine, Houston, TX 77030.
Baehr
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D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D000941:Antigens / Q000378:metabolism; D019393:Arrestin; D001483:Base Sequence; D003001:Cloning, Molecular; D005136:Eye Proteins / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D066298:In Vitro Techniques; D051379:Mice; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D011994:Recombinant Proteins / Q000378:metabolism; D015293:Transducin / Q000378:metabolism
D000941:Antigens; D019393:Arrestin; D005136:Eye Proteins; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D006160:Guanosine Triphosphate; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D015293:Transducin