Expression in bacteria of functional inhibitory subunit of retinal rod cGMP phosphodiesterase.

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Abstract
The cGMP phosphodiesterase of vertebrate retinal rod outer segments plays a key role in visual transduction. A functionally active form of the inhibitory .gamma. subunit of the phosphodiesterase, which keeps the enzyme inactive in the dark, has been obtained in high yield from a synthetic gene expressed in Escherichia coli. A DNA sequence encoding the 87-residue bovine .gamma. subunit was chemically synthesized and assembled from 10 oligonucleotides. The synthetic gene was cloned into an expression vector that uses the promoter PL of .lambda. phage. E. coli was transformed with this vector, which encodes a fusion protein consisting of the first 31 residues of the .lambda. cII protein, a 7-residue joining sequence that is specifically cleaved at its C-terminal end by clotting protease factor Xa, and the 87-residue .gamma. subunit. The fusion protein was solubilized in 6 M urea and purified by ion-exchange chromatography on a CM-Sephadex column. The typical yield was 1 mg of fusion protein per liter of bacterial culture, which corresponds to the amount of .gamma. in about 2500 bovine retinas. Proteolytic cleavage of the fusion protein by factor Xa released a synthetic .gamma. with the same amino acid sequence as that of native .gamma.. Both fusion protein and synthetic .gamma. inhibited trypsin-activated phosphodiesterase with high affinity (Kd < 100 pM). Likewise, both were as effective as native .lambda. in inhibiting transducin-activated phosphodiesterase in rod outer segment membranes. This inhibition was reversed by the activation of additional transducin. Thus, the N terminus of .gamma. is not intimately involved in interactions with either the catalytic subunits of the phosphodiesterase or the activated form of transducin. In contrast, a C-terminal deletion mutant terminating at residue 74 of .lambda. stimulated rather than inhibited the trypsin-activated enzyme. Thus, the C-terminal region of .gamma. is critical for inhibition of the phosphodiesterase.