Abstract 1715 Pull-down analysis of MS2 L-protein

MS2 is a single-stranded RNA bacteriophage that infects Escherichia coli. In the final stage of the viral life cycle, the host bacterium is lysed using MS2 lysis protein, L. L is encoded by a 225-nucleotide gene producing the 75 amino acid protein. Data shows that expression of L from a plasmid in the absence of other viral proteins is sufficient for cell lysis. An S49C mutation or a C-terminal deletion of L blocks lysis activity. L's lysis mechanism is unknown, however, it is hypothesized that L may bind to a host factor within E. coli as part of the process. We will attempt to identify the ligand L binds and examine its potential role in host lysis. Glutathione S-transferase (GST) is a protein encoded by pGEX vectors that serves as an affinity tag to purify recombinant proteins. The GST tag is positioned upstream of L and serves as a handle for a pull-down assay to obtain the L-protein ligand complex. We will monitor an IPTG induction of cells harboring a pGEX vector encoding the fusion protein, GST-MS2-L for lysis activity. We will directly compare the wild-type GST-L fusion with GST fusions of two L mutants, S17C and S49C as positive and negative controls respectively. Upon confirmation of lysis, glutathione resin will be used to bind the GST-L protein-ligand complex. We will then electrophorese and stain the protein-ligand complex to identify unique proteins found in the wild-type GST-L compared to control GST-only and GST-L mutants. After these unique bands are extracted, their identity and their relationship to host E. coli will be explored to determine the function of L.