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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
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)
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
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