Download 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

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