Construction of plasmids to express different membrane proteins in E. coli
The detection and conversion of olfactory stimulus starts when odorants or pheromone molecules (small hydrophobic molecules) penetrate through the pores on the wall of sensillum into the lymph fluid, where they interact with the odorant binding proteins (OBPs) which carry these molecules to an odor-specific receptor and its common co-receptor Or83b (in Drosophila) complex present in the membrane of olfactory sensory neurons (OSNs), which detects the odorant. In OSNs, it is assumed that the binding between OBPs-odorants and receptor & co-receptor complex results in a signal transduction and further cation influx into the sensory neuron and depolarization of the cell.
But the molecular processes and the spatial arrangement between OBPs carrying odorants and its specific receptor and common co-receptor subunits are unknown. Distance related measurements between specific sites on the receptors and OBPs could be made in live cells using luminescence resonance energy transfer (LRET) using a genetically engineered lanthanide binding tags (LBTs). But, LBTs never have been used before in live cells. Thus, in order to calibrate future LRET experiments, we designed and constructed a test protein eDHFR-ANK-LBT (DAL). We fused the DNA coding for DHFR, ANK and LBT together using PCR yielding DAL. Later we expressed this protein in E. coli and purified that protein using Ni-NTA column. Using this purified protein and live E. coli cells expressing this protein, we performed binding assays, emission and luminescence studies which showed that this protein can be used as a model in LRET techniques.
We tried to express and purify the odorant binding protein LUSH which is present in the sensillar lymph of Drosophila melanogaster. LUSH detects the pheromone cis-11-vaccenyl acetate (cVA) and may interact with the co-receptor OR83b which we also tried to express and purify. We were successful in expressing the LUSH gene obtained from Dr. David Jones but we were unable to purify it using cation and anion exchange column except for some high M.W. impurities. Partially purified LUSH was then further used for performing binding studies.
Later, we synthesized and tried to express the co-receptor for LUSH, Or83b gene. We synthesized a codon-optimized gene for expression in E. coli, but were unable to express eOr83b. We truncated the Or83b and constructed eOr83b3Tm and LOOP3TM variants. We were unable to express these variants as well, we then decided to fuse these constructs with other proteins like DHFR, MISTIC and the native E. coli membrane protein succinate dehydrogenase and tried to express them. But we were unable to do so. For expression trials, we used these constructs pET and pBADCONTROL plasmids, but neither of them successfully expressed the protein.