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Vaccinia Virus G1L Protein Expression and Purification

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

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Vaccinia Virus G1L Protein Expression and Purification

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  1. 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

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

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

  10. Design G1L primers KpnI G1LPLEX5’ PCR G1LPLEX3’ SphI G1L Blunt end cloning pCR4 (amp)

  11. Generate G1L plasmid SphI SphI KpnI KpnI G1L 4xCRR 4xCRR Restriction digest Rgg/gtfG pCR4 (amp) PLEX (chlor/eryth) G1L Rgg/gtfG Rgg/gtfG G1L + Ligate PLEX (chlor/eryth) PLEX (chlor/eryth)

  12. Transform bacterial cells G1L Rgg/gtfG PLEX (eryth) Streptococcus gordonii GP 251

  13. G1L? (approx. 45kD) 75kD 50kD 25kD G1L-PLEX No plasmid 4xCRR-PLEX 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 secreted into media

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

  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

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