| The study of insect olfaction may one day help control pest insects. We used Drosophila melanogaster as a model organism to study insect olfaction. Pheromones/specific odorants are chemical signals through which the insects mate, aggregate, etc. In Drosophila, the olfactory receptor complex, which involves detecting the pheromone cis-11-vaccenyl acetate, has a receptor Or67d and co-receptor Or83b ubiquitously present in the fly, another common necessary receptor SNMP (Sensory Neuron Membrane Protein) as well as an odorant binding protein (OBP) called LUSH. Our research involved cloning, expressing and purifying LUSH. We attempted some six strategies to express this protein. The last strategy gave us our protein. The strategies were expression in pET21a vector, expression in pkLAC1 vector, expression using Green Fluorescent Protein as a fusion partner with LUSH, expression using synthetic gene technology, expression by making some minor modifications and finally, expression using the pET13a vector. After, successfully expressing in pET13a, we scaled up the protein production and did some four purification trials. We found a purification technique involving three phase partitioning followed by concentration, dialysis, vacuum filtration, low and high speed centrifugation, CM Sepharose Cation Exchange Chromatography and if needed Gel Permeation. After achieving a good level of purity (though not absolute purity), we did binding assays of LUSH with the fluorescent probe N-Phenyl-1-Napthylamine (1-NPN) to determine its binding constant and its conformational stability. The last step is to validate the pull down assay with cVA. |
