Expression of mouse rod photoreceptor cGMP phosphodiesterase γ subunit in bacteria

Abstract

We expressed the γ subunit of mouse rod photoreceptor cGMP phosphodiesterase (PDE) in the bacterial pGEX-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 PDEγ, equivalent to the PDEγ content of approximately 200,000 mouse retinas. Both the fusion protein and the cleaved PDEγ, to which a short kinase domain remains attached, are biologically active, inhibiting activated PDE in a manner comparable to native PDEγ. Immobilized PDEγ binds transducin α subunit charged with GTP, PDE α and β subunits, and, unexpectedly, arrestin (S-antigen).