Studies on proteins of the bovine retina through expression cloning

Mukund Manohar Paidhungat, Purdue University

Abstract

The interaction of rhodopsin with the G-protein transducin initiates the visual transduction cascade in the rod outer segments of the vertebrate retina. To study the opsin-transducin interaction, inducible expression systems were used to attempt expression of bovine opsin c-DNA in E. coli in anticipation of mutagenesis studies of potential opsin-G-protein interaction domains. The ultimate goal of these experiments was to combine a mutagenised opsin with native transducin to observe differences in activity. However, our results indicated that expression of opsin in E. coli is potently cytotoxic to the bacterial host before any significant accumulation of the recombinant opsin. A full length c-DNA for rod outer segment transducin (G$\sb{\rm t}\alpha\sb{\rm r}$) was isolated from a bovine retinal c-DNA library to attempt expression of this globular protein in E. coli, to serve as the variable component in the interaction system, instead of opsin as originally intended. During the screening of the library, the c-DNA for a novel member of the G-protein family was isolated and sequenced. The sequence of the protein deduced from this c-DNA has a high percent identity to the G$\sb{\rm i}$ sub-family and we have hence designated this protein G$\sb{\rm i}$ret$\sb\alpha$. The c-DNA's for G$\sb{\rm t}\alpha\sb{\rm r}$ and G$\sb{\rm i}$ret$\sb\alpha$ were expressed in E. coli as fusion proteins by using a plasmid vector, that efficiently expresses the E. coli cytoplasmic ribose binding protein A (rbsA), as a vehicle. The G$\sb{\rm t}\alpha\sb{\rm r}$/rbsA and G$\sb{\rm i}$ret$\sb\alpha$/rbsA fusion proteins isolated from the bacterial host were used to immunise laying hens. Antibodies of high specificity capable of recognising native transducin in retinal fractions were obtained from animals injected with the G$\sb{\rm t}\alpha\sb{\rm r}$/rbsA fusion protein. The antibodies specific for the G$\sb{\rm i}$ret$\sb\alpha$/rbsA fusion protein have not been demonstrated to recognise a candidate corresponding to native G$\sb{\rm i}$ret$\sb\alpha$ in the retinal fractions examined, possibly due to much lower amounts of this protein in the retina as compared to transducin. The technique of raising antibodies to recombinant fusion proteins capable of discriminating between highly related members of a protein family has been demonstrated.

Degree

Ph.D.

Advisors

Applebury, Purdue University.

Subject Area

Molecular biology

COinS