ASU TVDC Progress Report
01/29/08
Kathryn F. Sykes and Stephen A. Johnston
Completed Milestones: 25 and 32, 33
Active Milestones: 26, 28, 34, 35
Currently Inactive Milestones: 30, 36-38
Slide 1
MILESTONE 26
Prepare a highthroughput protein
production system
Gray: (sub )milestone title
Red: completed or inactive
Green: in progress
Test ORF synthesis
and select expression
constructs
Select and test
IVT Protocols
Select and test protein
purification protocols
Completed.
Expression templates
for prokaryotic expression
are optimized
Completed.
High yield IVT protocols
are optimized
Alternative to His/Ni
purification scheme
has been identified
Slide 2
Milestone 26
ML26-1 – completed
ML26-2 – completed
ML26-3 – Will close 03/01/08
a. Evaluate feasibility of the IVT depletion approach.
b. Evaluate feasibility of switching from an E. coli to
a wheat germ or rabbit reticulocyte IVT systems
c. Generate IVT polypeptides and related reagents
for optimizing UNM’s antigen-screening T cell
assay.
Slide 3
ML26-3. Previous Status
a. A set of size-fractionated IVT samples were
generated at ASU and tested at UNM for the
presence/absence of the cross-reacting E. coli
antigen in the IVT lysate. Filter units with 100 kDa
size cutoffs successfully eliminated the crossreactive components.
b. A set of eukaryotic IVT templates were generated
and shipped to UNM, however IVT reactions are
on hold only as backups for the E. coli system.
c. Proteins for the final boost of genetically
immunized mice were generated and shipped to
UNM.
Slide 4
ML26-3a Current status
Macrocomplex stability
1 – fresh IVT, 37oC, 4hrs;
2 – fresh IVT, 100kDa fltd;
3 – fr/thwed IVT, 37oC, 4hrs;
4 – fr/thwed IVT, 100kDa fltd;
5 – fr/thwed IVT, 37oC, 4hrs;
6 – fr/thwed IVT, 50kDa fltd;
7 – fr/thwed IVT, 37oC, 4hrs;
8 – fr/thwed IVT, 30kDa fltd
Slide 5
Conclusions
•Prolonged incubation and shaking
does not disrupt large complexes
(macrocomplexes) in lysate.
•A Freeze/ thaw cycle does destabilize
macrocomplexes.
Slide 6
ML26-3a Current status
Recovery of newly synthesized polypeptide
post size-fractionation
1 – no template, 4hrs at 37oC;
2 – same as 1, 100kDa filtered;
3 – calm, w/out incubation;
4, 6, 8 – calm, 37oC, 4hrs;
5 – same as 4, 100kDa filtered;
7 – same as 6, 50kDa filtered;
9 – same as 8, 30kDa filtered
Commassie Stain
Slide 7
Autoradiograph
Conclusion
•Filtration enriches polypeptide in lysate.
•Nonetheless, filtration traps more than 50%
of the polypeptide products, even with the
100 kDa unit.
Slide 8
ML26-3a Current status
Effects of dilution and Tween20 on lysate
depletion
1 – IVT fltd w/out dilution
2 – 1/10 IVT w/out filtration
3 – fltd 1:10 dltd with Tween
4 – fltd 1:10 dltd w/out Tween
5 – 1/3 IVT w/out filtration
6 – fltd 1/3 dltd with Tween
7 – flt 1/3 dltd w/out Tween
Slide 9
Conclusion
•Filtration removes ~90% of total E. coli
proteins in lysate.
•Dilution of lysate sample or addition of
detergent has no significant effect on protein
depletion.
Slide 10
ML26-3a Current status
Effects of dilution and Tween20 on
product recovery
Recovery
ug
%
De novo protein
Slide 11
Ova
Ova
Ova
Ova
Ova
Ova
Ova
Ova
undiluted unfiltered
undiluted
filtered
1/10
unfiltered
1/10
filtered
1/10 Tween filtered
1/3
unfiltered
1/3
filtered
1/3 Tween
filtered
55.8
6.2
50.8
8.2
7.0
55.0
3.0
4.5
FTU1695
FTU1695
FTU1695
FTU1695
FTU1695
FTU1695
FTU1695
FTU1695
undiluted unfiltered
undiluted
filtered
1/10
unfiltered
1/10
filtered
1/10 Tween filtered
1/3
unfiltered
1/3
filtered
1/3 Tween
filtered
49.4
8.4
60.9
11.8
12.6
49.9
5.6
4.5
11.1%
16.1%
13.8%
5.5%
8.2%
17.0%
19.4%
20.7%
11.2%
9.0%
Conclusions
•A 10-fold dilution of IVT reactions MAY
modestly improve recovery.
•Significant losses are associated with all
filtrations.
Slide 12
ML26-3a Current status
An alternative: acetone precipitation
Autoradiograph
BSA
1695
GFP
Slide 13
40%
50% 90%
20% 30%
S
40%
50% 90%
P
S
P
S
P
S
P
S
30%
P
S
P
S
P
S
P
S
P
S
20%
Coomassie
Conclusion
•A 40% acetone “cut precipitates most of the large
E.coli proteins and/or complexes,
•Yet maintains 3 smaller proteins, <40kDa proteins,
in the supernatant.
Slide 14
ML26-3a Current status
His-tag based purification from acetone
fractionated IVT reactions
40%
M
E
P
S
F
E
P
S
F
E
35%
P: acetone precipitate E: eluted off Ni beads M: marker lane
S: acetone soluble
F: flow through, Ni beads
Slide 15
Conclusion
•Acetone cut enables very high efficiency purification
of affinity-tagged product from lysate.
•Same problem of significant sample losses (50%) is
associated with purification from acetone depleted
IVT reactions.
•This is no more or less than losses also observed
with purified recombinant proteins.
Slide 16
ML26-3. Next steps
• A set of acetone fractionated control
polypeptides have been shipped to UNM for Tcell analysis…
• If results show removal of cross-reactive
component, yet antigen-specific stimulation,
we will implement acetone fractionation as a
replacement for filtration based depletion.
• Generate a set of IVT synthesized, acetone
fractionated FTU proteins and ship these to
UNM
Slide 17
MILESTONE 28
Build SCHU S4
proteome
Gray: (sub)milestone title
Red: inactive
Green: in progress
Build ORF expression
library corresponding
to proteome
Generate complete
protein-fragment library
Array protein-fragments
into measurable pools
For T cell stimulation
Active
On-hold
Inactive
Slide 18
ML28-1. Current Status
SCHU S4 ORF library has been initiated
but expression constructs are on hold
until final protocol for the T cell assays
is decided.
Slide 19
ML28-1. Next steps
• Initiate protein library production
Slide 20
MILESTONE 34
Pilot studies of optimization of RNA
isolation and hybridization conditions
Gray: (sub )milestone title
Red: completed
Green: in progress
RNA Isolation (UNM
Hybridization Conditions
Initial testing of heavily infected lungs
Perform CFU analyses and compare with purified RNA
Testing Maui Hybridization chamber
Amplification testing of Schu S4 RNA
with and without mouse lung RNA
RNA isolated from infected lungs received from UNM
Slide 21
MILESTONE 35
Array hybridations with mouse RNAs
from virulent Schu 4 infection
& RT PCR confirmation of candidates
Slide 22
Gray: (sub )milestone title
Red: completed
Green: in progress
Virulent Schu 4 Samples
RT-PCR Confirmations
Initial samples
Dose-Response of Infection
To Be Determined
Raw Signal Intensity
Histograms of Microarrays of
Amplified SCHU S4 RNA
Challenge dose 101
mg amplified
Slide 23
74
102
103
104
105
106
107
98
98
101
112
62
35
Spearman Correlations of Microarray
Data Between Challenge Dose
10^2 10^3 10^4 10^5 10^6 10^7
10^1 0.645
10^2
0.738
10^3
0.758
10^4
0.739
10^5
0.495
10^6
0.135
Slide 24
Spearman Correlations of Microarray
Data Between Experiments
Experiment 2
Experiment 1
10^3 10^4 10^5 10^6 10^7
10^3 0.618
10^4
0.616
10^5
0.644
10^6
0.423
10^7
0.127
Slide 25
Comparisons of Genes Amplified from
4 Hour and 4 Day Infections
103
104
4 Hours
78
103
59
293
28
254
68
109
105
Slide 26
4 Days
39
247
Conclusions: slides 23-26
• This dose response experiment repeats
results seen in the first experiment.
– Good correlations between challenges of
103-105
– High dose animals do not correlate as well
• There may be some early regulated
genes detectable (4 hrs) as compared to
late infection gene expression (4 days).
Slide 27
Upcoming Transcriptome Goals
• Q-PCR validation of the hits
• Additional genomic normalization studies
• Establish next experimental parameters for MS35
•Time Course Experiment
•Challenge with 103 SCHU S4 organisms
•Harvest 1,3,5,7 and 24 hours
•Parallel cultures in Chamberlain’s medium
Slide 28
Action Items
• Mitch will repeat microarray experiments with RNA from
4 hr 10e6 and 10e7 SCHUS4 infected mouse lungs and
will also try diluting the samples to dilute out a
contaminant.
• can you take acetone precipitate supernatant and run
over a 100kda filter to look at yield/ purity? Action: Kathy
thinks ASU has done this and will add to Tech report
next month.
• Kathy- ASU will be sending the acetone precipitation
purified proteins this week (1/29) to Terry for cell assays.
• Terry and Mitch look at the Agilent profiles….Could the
prokaryotic rRNAs (16s and larger s) be seen on the
total RNA profiles in the 10e6 to 10e7 infection?
Slide 29