Modelling, Cloning, and Expression of the J domain of C. elegans Rme-8 Protein

Stephen F Austin State University, Baker Pattillo Student Center, Student Center Theatre and Twilight Ballroom

Rme-8 is a J domain containing plasma membrane protein that is required for endocytosis in various cells The J domain is a characteristic structural motif found mainly in heat shock protein 40 (Hsp 40 or DnaJ) and other proteins such as Rme-8 Within the J domain is a tripeptide, the HPD motif, that is required by the J domain protein to interact with and stimulate the ATPase activity of Hsp70 a major cellular chaperone Rme-8 protein in C elegans CeRme-8 has not been identified with a particular Hsp70 partner CeHsp70 1 is the only cytosolic Hsp70 in C elegans therefore, we hypothesize that it is the binding partner for the J domain of CeRme-8 To test this hypothesis, we first need to express and purify the J domain of CeRme-8 We report herein the successful cloning and expression of the J domain of CeRme-8 Computer modelling revealed that the amino acid sequence of the J domain of CeRme-8 folds into the canonical J domain conformation, containing the HPD tripeptide Complementary DNA of the J domain of CeRme-8 was cloned into the pGEX-Tev-KG plasmid, in frame with the gene for glutathione S transferase ( to yield a GST CeRme-8 fusion protein IPTG induced expression of the expected 37 kilodalton fusion protein was confirmed by both SDS-PAGE and western blotting using antibody against GST Work is ongoing to develop a protocol for purification of both GST tagged and untagged J domains of CeRme-8 Future work will involve testing the effect of the J domain protein on the ATPase activity of CeHsp70-1