ASU TVDC Progress Report
11/27/07
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
or inactive
Red: completed
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 approaches
are under cont. evaluation
Slide 2
Sub-Milestone 1
Test ORF synthesis and
select expression constructs
Slide 3
Sub-Milestone 1. Current Status
• Development, testing and optimization of IVT template for
bacterial expression system are as complete as possible at
this point. We have generated sufficient amounts of
synthetic promoter and terminator fragments to complete the
entire program. Single batches should maximize sample
consistency.
• As we await results of the ongoing validation experiments for
the bacterial-lysate produced polypeptides in T cell assays,
we have been developing alternative expression cassettes for
eukaryotic in vitro expression systems.
• Namely, we have rabbit reticulocyte and wheat germ based
IVT systems as alternatives. Expression efficiency of these
alternative cassettes is not yet tested. Expected efficiency is
at least 100 times lower than that of the prokaryotic cassette
system.
Slide 4
Sub-Milestone 1. Conclusions
• As originally planned sub-milestone 1 is
complete.
• If necessary, we are prepared to switch to an
eukaryotic expression system, although
significant yield reduction is expected.
• If eukaryotic system is selected, additional
time will be needed to optimize.
Slide 5
Sub-Milestone 2
Select and test IVT protocols
Slide 6
Sub-Milestone 2. Current Status
• IVT condition has been optimized. An
adequate substitute for the ProteoMaster
machine (Roche) has been found. HiGro
provides sufficient agitation and handles
32 96-well plates at the time.
• Developed HTP protocol reliably
generates > 25ug of protein per reaction.
Slide 7
Sub-Milestone 2. Conclusions
• We are ready for HTP protein production in E.
coli based IVT systems.
• If the E. coli system is found unacceptable in
T cells assays, then the eukaryotic systems
can be further evaluated with regard to:
performance in the T cell assays, and
generating sufficient quantities of material.
Slide 8
Sub-Milestone 3
Select and test protein
purification protocols
Slide 9
Sub-Milestone. 3 Previous Status
• Efficiency of our His tag based protein
purification is highly comparable with those
reported by others with regard to loses during
purification (from 50 to 100%) and variability in
product purity (from 0 to 90%).
• Identifying required sample amount and purity
are under evaluation by the UMN team.
• Analysis of published data did not identify a
purification basis that performs better than the
His/Ni approach in use.
Slide 10
Sub-Milestone. 3 Current status
• Cross-reactivity.
• Removal of >80kD fraction from IVT reaction did not
eliminate the cross-reacting component(s).
• Assisting UNM in developing T cell assay for screening the
proteome for immunogens.
• pCMVi-LS-OVA, -groES (FTT1695), -groEL (FTT1696), IglC2 (FTT1712c), katG (FTT0721c), -Tul4 (FTT0901) have
been made and confirmed. Genetic immunization
scheduled to start 11/28/07.
• The same set of genes was cloned for recombinant E. coli
expression (in vivo). Large scale protein expression and
purification is in progress. Proteins will be shipped to
UNM for immunization (boost) material and for antigen in
specific T cell immunoassays. Optimizations have been
performed (See figure…)
Slide 11
Recombinant protein preps
1
Legend:
1: ladder
2: FTU 1695_A, 37C, 2 hrs, 0.5M IPTG
3: FTU 1696Aa 37C, 2 hrs, 0.5M IPTG
4: FTU 1696Bb 37C, 2 hrs, 0.5M IPTG
5: FTU 1696Bb 37C, 4 hrs, 0.1M IPTG
6: FTU 1696Bb 30C, 4 hrs, 0.5M IPTG
7: FTU 1696Bb 37C, 3 hrs, 0.5M IPTG
8: FTU 1696Bb 37C, 4 hrs, 0.5M IPTG
9: FTU 1696Bb 42C, 4 hrs, 0.5M IPTG
Slide 12
2
3
4
5
6
7
8
9
Sub-Milestone 3. Conclusions
• We will proceed with LEE assembling and protein
production as soon as minimal requirements for
polypeptide amount and purity are defined.
• To define these parameters an adequate test model
is needed. We have proposed to use T-cells isolated
from OVA/LVS co-immunized mice and also a
selected set of FTU-ORF immunized animals.
• All constructs needed for immunization are now
available. Immunizations start tomorrow.
Slide 13
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
On-hold
awaiting final decision
on LEE format
Inactive
Inactive
Slide 14
Sub-Milestone 1
Build ORF expression
library corresponding to
proteome
Slide 15
Sub-Milestone 1. Previous Status
• ORF amplification protocols are in place.
• Templates and primers for promoter and terminator
amplification have been tested and readily available.
• Database management and tracking system is in
place.
• HTP ORF production has been tested. PCR success
rate is 99%.
Slide 16
Sub-Milestone 1. Current Status
Building SCHU S4 ORF expression
library is on hold until the T cells crossreactivity and specificity issues are
resolved.
Slide 17
Proteome Project Goals for Next
Month
• Continue to work with Drs. Terry Wu and Rick
Lyons in developing ideas and testing them to push
through this T cell assay antigen hurdle.
• We are involved in testing new synthetic eukaryotic
promoters. These are aimed at providing higher
levels of expression from eukaryotic cell-free
lysates. The expression systems are early stage
technologies but might be employed --if necessary.
Slide 18
MILESTONE 33
Printing and testing
GDP confirmed
Printing arrays
Comparisons of substrate
Poly-L Lysine vs. Corning Ultragaps
Compare TIGR PFGR Arrays to in house arrays
RNA shipped 1/29/2007
Slide 19
Gray: (sub )milestone title
Red: completed
Green: in progress
GDP Confirmation
Testing of linear amplification of prokaryotic
Transcripts (LAPT) process and dilution testing of
Schu S4 RNA with and without mouse lung RNA
Microarray Signal Intensities
Raw Signal Intensity
ASU v TIGR
ASU Slide 1
Slide 20
ASU Slide 2
TIGR Slide 1 TIGR Slide 2
Raw Signal Intensity
Reconstitution Experiments
1 mg
Slide 21
0.5 mg
0.1 mg
0.05 mg
0.01 mg
0.001 mg
0.0001 mg
0.00001 mg
Spearman Correlations Between Samples
SCHU S4 RNA in mg
1
Slide 22
SCHU S4 RNA in mg
0.5
0.1 0.05 0.01 0.001 0.0001
0.5 0.764
0.1
0.05
0.01
0.001
0.0001
0.00001
0.756
0.776
0.751
0.898
0.794
-0.007
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 23
Hybridization of Single Amplified Fragments to Microarrays
FTU0242A
FTU0457A
FTU0580A
FTU0713B
FTU0890A
FTU0897A
FTU0988A
FTU1295B
FTU1365B
FTU1658A
Slide 24
Exp 1
ASU Array
Y
Y
Y
N
Y
N
Y
Y
Y
Y
Exp 2
ASU Array
TIGR
Y
N
Y
N
Y
Y
N
N
MILESTONE 35
Array hybridations with mouse RNAs
from virulent Schu 4 infection
& RT PCR confirmation of candidates
Slide 25
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
Normalized vs. Raw Signal Intensities of
Individual Samples
Normalized
Slide 26
Raw
Heatmap Comparisons of Averaged vs.
Individual Sample Results
Color map arranged by individual dataset
Slide 27
Color map arranged by pooled dataset
Correlation of Normalize Signal Intensities
Between Pooled and Averaged Data
107
Averaged data
103
Spearman = 0.58
Slide 28
Pooled Data
Spearman = 0.84
Upcoming Transcriptome Goals
•
•
•
•
Slide 29
Q-PCR validation of the hits
Additional TIGR to ASU microarray comparisons?
Extended dose-response in vivo challenge
Establish next experimental parameters for MS35
Semi-Annual QA report
1/1/2007 – 6/30/2007
Item
QA performed by
Schedule for
testing
Last tested
Microarray spotters Contractors to ASU Yearly/as needed
and readers
1/26/2007
Pipetmen
Contractor to ASU
Yearly
05/2007 or new
Refrigerators/
Freezers
ASU-REES
Surveillance
Daily
6/30/2007
Safety Eye wash
Biodesign/ASU
Institutional Safety
Weekly
6/29/2007
Slide 30
Action Items
•
•
•
•
•
•
•
•
Kathy will email ATP wash protocol to Terry asap.
Can Terry verify that retic or wheat germ ivt will be cleaner in the T cell
assays than e coli ivt reactions? If retic and wheat don’t work better then
really need to push on E coli.
Alex will prepare translations with and without template with retic and
wheat germ ivt systems for UNM
Terry will wash bead bound IVT proteins with ATP and put into T cell assay;
splitting the sample (with and without ATP wash) for comparison, to reduce
E coli protein crossreactivity in Elispot assay for T cell responses
Alex: start making 1 FTU and non relevant protein translation, making
several reactions for Terry for testing further.
UNM and ASU need to plan the next mouse dose response infection
experiment so UNM can provide more RNAs to ASU for microarray gene
expression studies.
MS completion timelines- end of next week for Kathy 12/7 and end of this
week for Mitch 11/30.
Next two ASU technical calls: 12/19/07 Wed noon-1pm MT and 1/29/08
Tuesday noon-1pm MT
Slide 31