Vaccinia Virus G1L Protein Expression and Purification Emily Williamson HHMI Summer Undergraduate Research Program 2004 Laboratory of Dr. Dennis Hruby Oregon State University Vaccinia virion Vaccinia Virus Relative of the Variola virus, the causative agent of smallpox Can be used as a model system to develop antismallpox drugs and vaccines G1L is a potential drug target G1L is essential for viral maturation Role in maturation G1L is believed to be responsible for steps in morphogenesis of virion between DNA condensation and infectious oval virion formation Infectious oval virus G1L not expressed G1L is a potential drug target G1L is essential for viral maturation The protease G1L is a promising target for antiviral drugs because of their lack of homology to human enzymes Similarity of G1L to other proteins HXXEH motif (active site) 100% Vaccinia 98% Variola 99% Camelpox 99% Monkeypox 28% ymxG (Hypothetical zinc protease from B. subtilis) Black lines: Lack of homology G1L is a potential drug target G1L is essential for viral maturation The protease G1L is a promising target for antiviral drugs because of their lack of homology to human enzymes Purified G1L can be used to screen for effective antiviral drugs against smallpox How to overexpress the G1L protein PLEX (plasmid-borne expression): a protein expression vector system that uses Streptococcus gordonii to produce and secrete a protein (G1L) Multiple plasmids are expressed in cells Regulated by S. gordonii promoter Signal sequence fused to N-terminus of G1L PLEX Plasmid Promoter and signal sequence Erythromycin Resistance (S. gordonii) Original system developed by Travis Warren Gene of interest Chloramphenicol Resistance (E. coli) Design G1L primers KpnI G1LPLEX5’ PCR G1LPLEX3’ SphI Blunt end cloning Generate G1L plasmid KpnI SphI KpnI SphI Restriction digest + Ligate Transform bacterial cells Streptococcus gordonii GP 251 Is G1L Secreted from S. gordonii? Reducing SDS-PAGE, silver stain analysis of proteins in supernatant of S. gordonii cultures containing G1L-PLEX plasmid G1L? (approx. 45kD) 75kD 50kD 25kD Immunoblot of G1L-expressing colonies with α-FLAG HRP antibody G1L secreted into media 4xCRR-PLEX No plasmid G1L-PLEX How to purify G1L Grow S. gordonii culture Centrifuge S. gordonii culture Pour supernatant into Chromatographic column To isolate G1L Since G1L is secreted out of S. gordonii into the media, the supernatant is analyzed G1L protein Once expressed and purified… Make G1L-specific antibodies Analyze activity of G1L on various substrates Test various compounds on G1L activity in vitro Acknowledgements Howard Hughes Medical Institute Dr. Dennis Hruby Members of the Hruby Lab With a special thanks to Marika Olcott and Travis Warren