Vaccinia Virus G1L Protein Vaccinia Virus G1L Protein Expression and PurificationExpression and Purification

HHMI Summer Undergraduate Research Program 2004

Laboratory of Dr. Dennis HrubyOregon State University

Emily Williamson

Vaccinia virion

Vaccinia VirusVaccinia 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

G1L is a potential drug targetG1L is a potential drug target

G1L is essential for viral maturation

Role in maturationRole 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 targetG1L 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

Vaccinia100%

ymxG (Hypothetical zinc protease from B. subtilis)

28%

VariolaCamelpox

Monkeypox

98%

99%

99%

HXXEH motif(active site)

Black lines:Lack of homology

G1L is a potential drug targetG1L 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 How to overexpress the G1L proteinprotein

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 PlasmidPLEX Plasmid

Original system developed by Travis Warren

Promoter and signal sequence

Gene ofinterest

ChloramphenicolResistance (E. coli)

Erythromycin Resistance (S. gordonii)

Design G1L primersDesign G1L primers

KpnI

SphI

G1LPLEX5’

G1LPLEX3’PCR

Blunt end cloning

Generate G1L plasmidGenerate G1L plasmid

KpnISphI KpnI SphI

+

Restriction digest

Ligate

Transform bacterial cellsTransform bacterial cells

Streptococcus gordonii GP 251

Is G1L Secreted from Is G1L Secreted from S. gordoniiS. 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 plasm

id

4xCR

R-P

LE

X

G1L? (approx. 45kD)

75kD50kD

25kD

G1L secreted into media

How to purify G1LHow to purify G1L

CentrifugeS. gordonii culture

Pour supernatant intoChromatographic columnTo isolate G1L

G1L protein

Grow S. gordonii culture

Since G1L is secreted out of S. gordonii intothe media, the supernatant is analyzed

Once expressed and purified…Once expressed and purified…

Make G1L-specific antibodies

Analyze activity of G1L on various substrates

Test various compounds on G1L activity in vitro

Acknowledgements Acknowledgements

Howard Hughes Medical InstituteDr. Dennis HrubyMembers of the Hruby Lab

With a special thanks to Marika Olcott and Travis Warren