ASU TVDC Progress Report
10/28/08
Kathryn F. Sykes and Stephen A. Johnston
Completed Milestones: 25, 26 and 32, 33, 34
Active Milestones: 28 and 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
Expression templates
for prokaryotic expression
are optimized
High yield IVT protocols
are optimized
Purification strategies are
developed
Slide 2
Last report :Protocol established for High yield,
High purity, HTP polypeptide production
T7
RBS
ATG
TRX
ORF
His
Term
Tosyl Magnetic beads for capture
IVT proteins
kD
250
150
100
75
50
37
25
20
15
Assembly of IVT LEE cassettes
In vitro translation of proteins
Slide 3
Problems solved
•
Highly variable rates of i) solubility and ii) loss of yield
occur during purification.
 Trx/His double fusion template
 full-length ORFs
•
Lysate carries one or more components that strongly
stimulate LVS immune T cells.




NHP cells,
Reconstituted IVT component mix,
Bead attachment via Trx tag
Co-translational bead binding.
Slide 4
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
for T cell stimulation
assays
Nearly complete
In process
Inactive
Slide 5
Testing large batch of NEB IVT kit
Kda
100
1
2 3 4 5 6 7 8 9 10 11 12
75
50
37
25
• 12 Linear expression templates were from FTU 384 primer mix
•(Long ORF 2 PCR plate 2 row A)
• 5 ul of total IVT reaction lysate from each sample were loaded
(evaluating
synthesis)
•IVT reactions were run w/o beads or purification
Slide 6
Testing large batch order of anti-TRX antibody
and beads (purification)
250
150
100
75
50
37
25
20
15
10
• Odd number: IVT protein eluted from beads (represents
bound/purified fraction)
•Templates were from FTU Long ORF 5 384 Primermix PCR plate 2
row A
• Even number: 1/5 reaction supernatant from beads (represents
unbound/unpurified fraction)
Slide 7
Testing storability of bead-bound
polypeptides
Kda
Ova
50
Beads
FTU721A
Supernatant
Beads
Supernatant
2
1
37
25
15
10
1
2
3
1
2
3
1
3
2
3
1: Immediately after IVT reaction was completed
2: Storage at 4oC, 5 days later
3: Storage at -80oC for 5 days, and thawed
Slide 8
Current status of LEE productions
1. We have assembled 2,065 high quality linear
expression element constructs of FTU ORFs,
which are ready for IVT reactions.
2. 128 ORFs ( from Long ORF 5 384-well plate)
and 36 ORFs( from Short ORF 2 384-well
plate) were not amplified. FTU primer
degradation during storage appeared to be the
problem.
3. To confirm, 12 FTU ORF primers from the
Short ORF 2 plate were reordered and tested
in amplification.
Slide 9
New primer testing
kb
1.5
1
2
3 4 5
6 7
8 9 10 11 12
1.0
0.5
• Primers for FTU 384 Short 2 plate 1 row A (A1-A12) were
reordered and used to amplify ORFs from SchuS4 genomic DNA
• All FTU ORFs were successfully amplified using the new primers
Slide 10
Next steps
• We have ordered oligos for the other
150 missing ORFs
• These last pieces of the library will be
made, assembled, and QA’ed
• We will communicate with Terry on
timing for initiating polypeptide
production and details of delivery
MILESTONE 35
Array hybridations with mouse RNAs
from virulent Schu 4 infection
& RT PCR confirmation of candidates
Gray: (sub )milestone title
Red: completed
Green: in progress
Virulent Schu 4 Samples
RT-PCR Confirmations
Initial samples
Dose-Response of Infection
Ongoing
Slide 12
Previous Status
• LAPT analyses have been performed on…
Two Dose Response Challenge Samples
103, 104, 105, 106, 107
101, 102, 103, 104, 105, 106, 107
1,3,5,7, and 24 hours post challenge
Second Time course experiment.. Ongoing
• qPCR established using 6 genes and reconstitution
samples.
Slide 13
Targeted genes for qPCR
FTT Designation Gene symbol
Gene Name
FTT0901
lpnA (Tul4)
conserved hypothetical lipoprotein
FTT0721c
katG
Peroxidase/catalase
FTT1712c
iglC2
intracellular growth locus, subunit C
FTT0548
dnaQ
DNA polymerase III, epsilon subunit
FTT0058
atpB
ATP synthase subunit
FTT0256c
Lipopolysaccharide protein
Normalization Control…
16S Ribosomal
MutS
Slide 14
qPCR (iglC to MutS)
Slide 15
Slide 16
Relative Expression of iglC to MutS
Sample
Ct SYBR MutS
Exp. 1 105
Ct SYBR
1712c
28.24
34.02
Expr. Level
SYBR
0.201
Exp. 1 106
25.23
32.27
0.484
Exp. 1 107
20.80
28.89
No Data
Used as
calibrator
Sample
Ct SYBR MutS
Exp. 2 104
Ct SYBR
1712c
31.39
34.65
Expr. Level
SYBR
0.102
Exp. 2 105
28.84
33.08
0.201
Exp. 2 106
25.70
31.28
0.510
Exp. 2 107
21.71
28.26
No Data
Used as
calibrator
Slide 17
LAPT Analysis
• We are having intermittent amplification failures
• Possible resolutions
• Kit problems
• Tested controls provided
• Inhibitors present in RNA samples
• Testing reconstitution samples
• Spike QC SCHU S4 RNA into sample RNA
Slide 18
Upcoming Transcriptome Goals
• LAPT
• Evaluate presence of inhibitor in current
sample set
• Re-establish amplifications with reconstitution
samples
• Q-PCR validation of the hits
• Find and evaluate new normalization gene for
relative quantification.
Slide 19
Action Items
•
•
•
•
•
•
•
Kathy is writing the MS #26 MSCR to send to UNM
UNM will communicate to ASU, regarding the date to produce the polypeptides for
testing with vaccinated NHP cells in vitro at UNM
UNM needs to determine how many polypeptides should be combined per well, prior
to ASU synthesis and arraying in plates
ASU will store the ORF primers in concentrated and not diluted form to maximize
primer stability
Barbara will ask Terry about the dates of the vaccinated NHP harvest, for the ELIspot
testing of ASU IVT products at UNM
Mitch will re-evaluate the 16s ribosomal protein as a qPCR control and try different
primer pairs
Mitch will keep UNM apprized of progress on reliability of the LAPT amplifications.
Slide 20