1. Vaccinia Virus G1L Protein Expression and Purification HHMI Summer Undergraduate Research Program 2004 Laboratory of Dr. Dennis Hruby Oregon State University Emily Williamson Vaccinia virion

2. Vaccinia Virus Relative of the Variola virus, the causative agent of smallpox Can be used as a model system to develop anti- smallpox drugs and vaccines

3. G1L is a potential drug target G1L is essential for viral maturation

4. 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

5. 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

6. Similarity of G1L to other proteins Vaccinia 100% ymxG (Hypothetical zinc protease from B. subtilis) 28% Variola Camelpox Monkeypox 98% 99% HXXEH motif (active site) Black lines: Lack of homology

7. 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

8. 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

9. PLEX Plasmid Original system developed by Travis Warren Promoter and signal sequence Gene of interest Chloramphenicol Resistance (E. coli) Erythromycin Resistance (S. gordonii)

10. Design G1L primers KpnI SphI G1LPLEX5’ G1LPLEX3’ PCR Blunt end cloning

11. Generate G1L plasmid KpnI SphI KpnI SphI + Restriction digest Ligate

12. Transform bacterial cells Streptococcus gordonii GP 251

13. Is G1L Secreted from S. gordonii? Reducing SDS-PAGE, silver stain analysis of proteins in supernatant of S. gordonii cultures containing G1L-PLEX plasmid Immunoblot of G1L-expressing colonies with α-FLAG HRP antibody G1L-PLEX No plasmid 4xCRR-PLEX G1L? (approx. 45kD) 75kD 50kD 25kD G1L secreted into media

14. How to purify G1L Centrifuge S. gordonii culture Pour supernatant into Chromatographic column To isolate G1L G1L protein Grow S. gordonii culture Since G1L is secreted out of S. gordonii into the media, the supernatant is analyzed

15. Once expressed and purified… Make G1L-specific antibodies Analyze activity of G1L on various substrates Test various compounds on G1L activity in vitro

16. Acknowledgements Howard Hughes Medical Institute Dr. Dennis Hruby Members of the Hruby Lab  With a special thanks to Marika Olcott and Travis Warren