OPTIMIZING EXPRESSION OF RECOMBINANT HIV FUSION PROTEIN IN E. COLI
Elena Gochez, Michelle E. McCully, William F. Degrado.
University of California, San Francisco, San Francisco, CA.
Since the beginning of the human immunodeficiency virus (HIV) epidemic in 1983, about 78 million people worldwide have been infected with the virus and 39 million people have died from infection. HIV infects and destroys the body’s CD4+ T cells, which are essential to the body’s ability to fight off disease. In HIV, the capsid that packs the viral genetic material is enveloped by a lipid bilayer containing surface proteins. Gp41 is the viral surface protein responsible for fusion of the viral and target cell membranes initiating HIV infection. Protein-mediated fusion is not fully understood because the complete post-fusion structure is unknown. This project tests and optimizes protein expression of gp41 and its parainfluenza virus homologue, F protein, for structural and biophysical characterization. Our experimental approach is to transform plasmids containing genes for gp41 and F protein into 3 strains of E. coli: BL21, C43, and LOBSTR. Expression was assayed using SDS-PAGE as a preliminary step and a western blot to confirm the presence of the proteins. The proteins of interest were tagged with a fluorescent protein, and expression was monitored over time via fluorescence spectrometry. In the future, the best-expressing proteins will be purified and used to characterize the fusion process and the post-fusion conformation. Obtaining the entire post fusion structure of gp41 will be beneficial as it is a drug target for fusion inhibitors. New fusion inhibitor treatments will stop the progression of the disease and reduce transmission.