ASU TVDC Progress Report
6/24/08
Kathryn F. Sykes and Stephen A. Johnston
Completed Milestones: 25 and 32, 33, 34
Active Milestones: 26, 28, 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
Purification strategies have
been identified
Slide 2
Milestone 26
ML26-1: Completed.
•
ORFs are efficiently generated using oligo and gene
designs established in ML #25
•
LEEs are specifically and efficiently assembled from
construct designs established in ML #25
ML26-2: Completed.
•
We observe improved yields of polypeptide from
linear template in a continuous-exchange cell-free
“feed” system
•
All components and parameters have been titrated to
optimize yield and minimize cost
•
Bacterial in vitro transcription/translation system is
optimized for HTP application.
Slide 3
ML26-3 : Develop protein
purification protocols
Slide 4
ML26.3 Previous Status
• Proper conformation appears to be critical for
efficient separation of cross-reacting molecules
from polypeptides by:
• size filtration
• acetone precipitation
• affinity (Ni-binding) purification
• Several folding-independent purification methods
have been developed
• Several methods to improve folding have been
developed.
• Several depletion methods have been developed
as an alternative to polypeptide purification
Slide 5
ML26-3. Update
Slide 6
Imidazole wash titration: LVS cross-reactivity of E.
coli proteins nonspecifically bound to Ni beads
No template
Ova
50% Naïve+ 50% DO11.10
20mM
50mM
100mM
200mM
500mM
20mM
50mM
100mM
200mM
500mM
LVS vaccinated
Mass spec analysis of bead-bound proteins showed similar
composition after either 20mM or 500mM wash
Slide 7
Conclusion
• Proteins are nonspecifically stuck to
Ni beads.
• Imidazole wash concentration has no
effect on cross-reacting material.
• Imidazole titration elutes ova
polypeptide as expected at ~100mM.
Slide 8
Pre-clear IVT lysates with anti-LVS
antisera
1
2
3
4
5
50% Naïve+
50%
DO11.10
LVS
vaccinated
LVS immunized sera incubated with protein G beads for 1 hour (load beads with antiLVS)
Beads were washed with PBS
E.coli lysate (pre-reaction) from IVT kit was incubated with IgG bound protein G beads
for another hour (capture cross-reacting E. coli proteins)
Supernatant from beads (anti LVS depleted material) was tested in T-cell assay
1: No pre clearing of IVT lysate, 2: 10ul, 3: 20ul, 4: 50ul, 5: 100ul protein G beads used
for pre-clearing
Slide 9
Conclusion
• Depleting IVT lysates of components that bind antiLVS antibodies reduces cross-reactivity.
• This result confirms that we are observing specific
cross-reactivity between Francisella and E. coli
antigens.
• However we are presumably depleting conserved
translation machinery
Slide 10
Testing NEB IVT kit for crossreactive components
• NEB pure IVT kit contains only essential components
for transcription and translation
• This limits the diversity of E.coli proteins exposed to
T-cells
Testing depletion of E. coli IVT
proteins post-reaction
• Removal of trxn/ranslational machinery after
polypeptide production will not interfere with synthesis
• However, any FTU polypeptide with homology to E. coli
will be depleted.
Slide 11
Post-IVT reaction depletion of E.coli
proteins
from
OVA
polypeptide
prep
1
2
3
4
1
2
3
4
50% Naïve+ 50%
DO11.10
LVS Vaccinated
NEB PURE
INVITROGEN
1: IVT as is
2: 10ul E.coli IgG bound protein G beads
3: 50ul E.coli IgG bound protein G beads
4: 100ul E.coli IgG bound protein G beads
• IVT synthesized Ovalbumin polypeptide was incubated with indicated amounts of
protein G beads with attached anti-E.coli IgG
• The supernatant was used in T-cell assay
Slide 12
Conclusion
• NEB pure appears to be similarly immunestimulating relative to standard lysate
composiiton
• High variability in samples
• This will be repeated with uniform bead
pipetting method
Slide 13
Sensitivity of Rabbit Retic IVT on Protein
G beads in T-cell assay
Ova
FTU901 FTU1695
Ova
FTU901 FTU1695
50% Naïve+
50% DO11.10
LVS Vaccinated
IVT as is
Protein G beads
Slide 14
Conclusion
• In positive control samples, the bead-bound
ova polypeptide stimulated a few more T cells
relative to .
• However, numbers are generally low (antigen
amount is low) and not discernable above
background in bead-bound IVT negative
control sample.
Slide 15
His/Thio-double tag LEE
Construct
ORF
T7-Pro
T7-Pro
6-His
Thio.
Thio.
ORF
6-His
T7-Term
T7-Term
N-terminal: Thioredoxin tag
C-Terminal: His tag
Slide 16
G-α-Thio
IVT thio fusion proteins bead bound via Thio or
His tag in T-cell assay
Ni
G-α-His
No
Template
No
Template
Ova
FTU901 FTU1695
50% Naïve+
50% DO11.10
Ova
FTU901
FTU1695
LVS Vaccinated
Slide 17
Conclusions
• The nickel NTA (nickel chelated
Nitrilotriacetic Acid ) is significantly more
“sticky”, with respect to the cross-reacting
lysate proteins, than either the anti-His or
Thio antibody.
• The His antibody is stickier than the Thio
antibody
• Use of the thio tag appears to be the a clean
approach (no or very low amounts of
nonspecifically bound IVT components.)
Slide 18
Set-up to test Sensitivity of an
antigen in the T-cell assay
• Recombinant ovalbumin and FTU proteins
were purified (bound and eluted) from affinity
column (Ni) and quantified
• Four different concentrations prepared from
stock and used for T-cell assay were
0.1ug/ml, 1.0ug/ml, 5.0 ug/ml, and 10ug/ml
(0.5ng, 5ng, 25ng, and 50ng)
• 100ul of the eluant was used to bind Ni
beads, Protein G beads with anti-his ab, or
left in the PBS buffer
Slide 19
Ova
1
2
3
4
1
2
3
FTU1695
4
1
2
3
4
PBS ProG Ni
FTU1695
FTU901
Purified antigen
FTU901
PBS ProG Ni
50% Naïve+ 50% DO11.10
R:\GeneVac\FTU\Contr
act\Proteome\FTU IVT
Data\FTU gels\FTU
HTP IVT Coomassie
gels
LVS vaccinated
1: 0.1ug/ml, 2: 1.0 ug/ml, 3: 5.0 ug/ml, 4: 10.0 ug/ml
Slide 20
Comparison of antigen (OVA) presentations in T-cell assay
250
PBS
Ni beads
Protein G beads
Number of spots
200
150
100
50
0
0.1
1.0
5.0
10.0
Antigen concentration (ug/ml)
Slide 21
Conclusions
• The protein G beads with attached
antibody provide higher sensitivity than
Ni beads in T cell assay.
• Our assay sensitivity is sufficient to
specifically detect ~5ug/ml of antigen
Slide 22
Next steps
1.
2.
3.
Generate magnetic beads with covalently attached
anti-Thioredoxin to increase antigen binding
capacity. Protein G beads have limited Fc binding
capacity.
Capture FTU IVT Thio-fusion polypeptides using
these beads (during and after rxn) and evaluate in
T-cell assays
Mice have been immunized with IVT lysate. We will
collect sera to use as reagent in post-IVT reaction
clearing step. IVT bound beads will also be used
to generate antisera reagent for depletion step.
This will reduce loss of FTU polypeptides with E.
coli homology. Cleared samples will be tested for
cross-reactivity in T-cell assay
Slide 23
Dynabeads M-280 Tosylactivated
•Hydrophobic, monodisperse magnetic
particles (2.8 m in diameter) with ptoluene-sulfonyl (tosyl) groups, further
surface activation is not required.
•Allows easy coupling of antibodies with
optimal orientation for affinity
purification of proteins.
•Covalent coupling to primary amino- or
sulphydryl groups in proteins/peptides
Slide 24
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
Preparing plan
Inactive
Slide 25
Next Steps
• The last protocol decisions with respect to
IVT sample clearing/purifying are becoming
independent of ORF and polypeptide
production.
• In parallel with optimizing the sample
preclearing method and finalizing bead
protocol for affinity tag binding, we propose
initiating library production
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
To Be Determined
Slide 27
Previous Status
•Two biological replicates of a dose response challenge
•Handled independently
•Positive correlation between dose responses
•Positive correlation between experiments
•Repeat amplifications were performed for reproducibility
studies
Slide 28
LAPT of in vitro grown SCHU S4
Slide 29
qPCR Assessment of FTT901 (Tul4)
Slide 30
FTT901 Standard Curve
Slide 31
Conclusions
•Problems with in vitro produced RNA
•Losses after RNAeasy purification
•Poor amplification
•Poor labeling
•qPCR Initial runs show good differentials
with genomic samples
Slide 32
Upcoming Transcriptome Goals
• Q-PCR validation of the hits
•Primers are designed now for 7 genes
• Time Course Experiment Repeat
•Rat and Mice
•Challenge with 104 SCHU S4 organisms
•Harvest 1,3,5,7 and 24 hours
•Parallel cultures in Chamberlain’s medium
Slide 33
Action Items
•
•
•
•
•
•
•
•
Terry will repeat the ELIspot assay again with the IVT thio fusion proteins bead bound
via thio or His tag or Ni
ASU/UNM could use 2 concentrations for each antigen in the ElIspot T cell assay to
show a dose effect over the background.
Rick wants ASU/UNM to perform the T cell ELIspot assay with NHP lymph node T
cells from vaccinated primates available in approximately 10 days, and then 35 to 42
days.
Alex and Terry: develop a written protocol for the use of the NHP lymph node cells
with many ASU IVT products purified by different approaches. Include the use of
lysates, different purification strategies and give the IVT’s to Terry to use in the NHP
hylar lymph node assays detected by Elispot
ASU can prepare the plan for making the LEES etc for the protein library generation,
but should not implement the plan yet.
Rick wants to see the next confirming experiment by ASU/UNM in the mouse one
more time, before starting the mass LEE production. Don’t implement the plan until
one more confirmatory mouse experiment is done.
Terry , send twice as much RNA from next SCHU S4 growth in Chamberlains to ASU
Next ASU tech call is 7/22/08 4th Tuesday in July. Monthly technical reports are due
on 7/7/2008.
Slide 34