Download Does the baculovirus sulfhydryl oxidase Ac92 associate with and

Taylor Bendt
Faculty advisor: Dr. Gary Merrill
 Important for cancer prevention
DNA Damage
p53
DNA repair
p21
Apoptosis
Cell cycle arrest
http://www.topnews.in/files/CancerCells22.jpg
Genome maintenance

If p53 is oxidized
Ac92 could have an
inhibitory function.
Prevent apoptosis
 Prevent cell cycle
arrest
DNA Damage
p53

Insights for preventing
cancer
DNA repair
p21
Apoptosis
Cell cycle arrest
Genome maintenance

A baculovirus is a viral parasite
that infects insects

Ac92 is a baculovirus protein
that binds FAD and has
sulfhydryl oxidase activity

Human p53 expressed from a
baculovirus vector copurifies
with Ac92 (Ac92 and human
p53 can physically interact)
http://www.biochem.wisc.edu/faculty/friesen/

Involved in viral assembly, as has been shown for
sulfhydryl oxidases of mammalian viruses

defeats the antiviral activity of host cell p53
(mechanisms to defeat p53 are common in
mammalian viruses)

Ac92 binds to Sfp53 (the p53 protein of
Spodoptera frugiperda – the natural host of
baculoviruses)

Ac92 oxidizes Sfp53

Sfp53 is inhibited by oxidation
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
Ac92 binds Sfp53: co-immunoprecipitation
assays
Ac92 oxidizes Sfp53: sulfhydrylhistochemistry
Sfp53 is inhibited by oxidation: target gene
activation in transfected insect cells



Screen for recombinant bacteria that
overexpress Sfp53 and Ac92 that contain
reciprocal epitope tags
Prepare purified epitope-tagged Sfp53 and
Ac92
Determine if HA-tagged Sfp53 coprecipitates
Ac92 and if Flag-tagged Ac92 coprecipitates
Sfp53

Dr. George Rohrmann provided BL21 (E. coli)
glycerol stocks




3 clones with HIS tagged HA tagged Sfp53
5 clones with HIS tagged Flag tagged Ac92
Clones also contained kanamycin resistance
Cells are grown in liquid medium containing
kanamycin to kill all non-transformed bacteria


The proteins Sfp53 and Ac92 should only be
produced in the presence of IPTG
Comparing induced and uninduced protein
products should confirm that the clones work
Old Ac92
Sfp53
i
i
66
45
i
Sfp53
Ac92
31
21.5
14
6.5
IPTG
66
45
M.W Markers
+
+
pET28a-sfp53 clone 2
pET28a-sfp53 clone 1
pET28a-sfp53 clone 1
pET28 – Ac92 (old)
pET28 – Ac92 (old)
+
pET28a-sfp53 clone 3
pET28a-sfp53 clone 2
-
pET28 – Ac92 clone 9
pET28 – Ac92 clone 9
pET28 – Ac92 clone 10
+
+
-
pET28 – Ac92 clone 12
pET28 – Ac92 clone 11
pET28 – Ac92 clone 10
pET28a-sfp53 clone 3
+ + +
Sfp53
Ac92
31
21.5
14.4
6.5
Conclusions:
1) Sfp53 clones are correct (they produce a 46-kD that is inducible by IPTG)
2) Ac92 clones are incorrect (they don’t produce detectable protein)
Subsequent sequencing showed the clones had a frameshift mutation.
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Grow a 500 mL culture
Break by sonication in the presence of protease
inhibitors
Clarify by centrifugation
Bind His-tagged proteins to TALON resin
Wash resin
Elute bound protein with imidazole
Monitor purification by SDS-PAGE, and
Coomassie staining
http://www.odec.ca/projects/2007/meng7l2/new_page_8.htm
135
95
72
52
42
34
26
17
10
1
2
3
4
5
6
7
8
9
10
250
150
100
75
Sfp53
50
37
25
20
15
10
Eluate 5
Eluate 4
Eluate 3
Eluate 2
Eluate 1
10 mM Imid.
Unbound
H.S Sup.
H.S. Pellet
Induced
Uninduced
M.W Markers
Eluate 5
Eluate 4
Eluate 3
Eluate 2
Eluate 1
10 mM Imid.
Unbound
H.S. Sup.
H.S. Pellet
Induced
Uninduced
M.W Markers
Sfp53
Conclusions: Most of the Sfp53 is contained in the high speed pellet, and relatively
little is contained in the purified portions.
By Lowry, the protein concentration of the most concentrated eluate
is less than 1μg/μL, not enough for use.


Not enough Sfp53
was purified.
May be trapped in
inclusion bodies.
http://aem.asm.org/cgi/content/full/69/2/1295

Grow bacteria in 18 ° after induction

Change induction time from 6 hours to 20

Known to work for Ac92 from previous work
250
150
100
75
Sfp53
50
37
25
Conclusion: The 18 ° growth did not help solubilize the protein, and no Sfp53
was purified.
Eluate 5
Eluate 4
Eluate 3
Eluate 2
Eluate 1
100mM Imid.
50mM Imid.
10mM Imid.
Unbound
H.S. Sup.
H.S. Pellet
Induced
Uninduced
MW Markers


A collaborator has managed to produce soluble
Sfp53 in BL21 cells
There is one difference in protocol: volume of
resuspension of the bacterial pellet before
breaking open cells by sonication
H.S. Pellet
H.S. Sup.
Resuspension
Induced
Uninduced
H.S. Pellet
H.S. Sup.
Induced
Uninduced
MW Markers
250
150
100
75
Sfp53
50
37
25
20
15
Conclusion: Even with larger resuspension volume Sfp53 is not solubilized.
Conclusion: The new Ac92 is
correct and produces a
detectible amount of protein
when induced.
Ac92 Clone 2
Ac92 Clone 2
-
+
-
+
Ac92 Clone 3
Ac92 Clone 1
+
Ac92 Clone 3
Ac92 Clone 1
-
Sfp53 Clone 3
Sfp53 Clone 3
IPTG
-
+
Ac92

The correctness of these
clones is also supported by
an orange color generated
when the protein binds to
TALON resin
250
150
100
75
50
Ac92
37
25
20
15
10
Conclusion: Ac92 was successfully purified using TALON RESIN.
Eluate 5
Eluate 4
Eluate 3
Eluate 2
Eluate 1
10 mM Imid.
Unbound
Clarified Lysate
H.S. Pellet
Induced
Uninduced
MW Markers



Receive Sfp53 from collaborator
Using co-immunoprecipitation detect
interaction
Test for oxidizing activity of Ac92 on Sfp53
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http://www.biochem.wisc.edu/faculty/friesen/
Dr. Gary Merrill
Dr. George Rohrmann
HHMI
Frances Cripps
URISC
Kevin Ahern