ASU TVDC Progress Report
8/11/08
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
Expression templates
for prokaryotic expression
further optimized
High yield IVT protocols
further optimized
Purification strategies have
been identified
Slide 2
MS26.1
•
•
•
•
Completed as originally planned.
ORFs are efficiently generated using
oligo and gene designs established in
MS #25
LEEs are specifically and efficiently
assembled from construct designs
established in MS #25
We identified methods to expand LEEs,
which were implemented this month.
Slide 3
26.1 Update
Slide 4
Tagging strategies for Linear Expression Template
T7
RBS ATG
T7
RBS ATG
T7
RBS ATG
T7
Bio
ORF
Term
His
ORF
Term
Thio
ORF
Term
ORF
RBS ATG
N-term tags
His Term
C-term tags
T7
RBS ATG
T7
RBS ATG
His
ORF
T7
RBS ATG
Thio
ORF
ORF
HA
His
His
Term
Term
Term
double-tag
two-tag
Slide 5
Length of LEE ~800bp
Cassette has approximately
800bp
Only His tag (213 bp) at the C
terminal
DNA ladder (bp)
2000
800
400
200
100
R:\GeneVac\FTU\Contract\Proteome\FTU IVT Data\FTU gels\FTU HTP IVT DNA gels\Other\FTU short plate
#1 amp2 7-23-07_02
Slide 6
Length of LEE~ 1200 bp
Cassette has approximately
1200 bp
ORFs ~500bp
Trx tag ( 417 bp) at the N
terminal
His tag (213 bp) at the C
terminal
DNA ladder (bp)
2000
800
400
200
100
R:\GeneVac\BAN\BAN IVT Data\BAN Thio LEE Amp Gel\BAN IVT Plate 01 Amp 7-9-08 02__
Slide 7
Length of LEE ~1500-1700 bp
Cassette has approximately
1500-1700 bp
ORFs ranges from 800-1000bp
Trx tag ( 417 bp) at the N
terminal
His tag (213 bp) at the C
terminal
R:\GeneVac\FTU\Contract\Proteome\FTU IVT Data\FTU gels\FTU HTP IVT DNA gels\Thio LEE Construct\Long Plate 1 Columns 1 and 2 LEE test 7-25-08
Slide 8
26.1 Conclusions
1.
2.
3.
4.
New designs enable size of LEEs to reach to 1500bp
LEE assembly is efficient in high throughput
formats
ORFs were assembled into templates of different
sizes and from different genomes with similar
success(F. tularensis and B. anthracis)
Yield of PCR products is ~3 ug
Slide 9
MS26.2
• Completed as originally planned.
• We observe improved yields of polypeptide
from linear template in a continuousexchange 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.
• We identified methods to improve IVT
system, which were implemented this
month.
Slide 10
26.2 Update
Slide 11
Robustness of cell-free polypeptide synthesis with
old vs. new LEE templates in high-throughput format
Slide 12
IVT production of a range of FTU polypeptide lengths
sizes is similarly efficient
1
2
3
4
5
6
7
8
9 10 11 12 13 14
MW of samples is on the next slide-
Slide 13
Calculated polypeptide yields are not dependent
on polypeptide length
Fragment Name
ORF Size
MW
#Ms
CPM
CPM Total
CPM #met
pmol prot
ug prot
1
FTU1484A
724
37,831
6
131186
787,116
131,186
348
13.18
2
FTU0504A
637
36,447
5
75096
450,576
90,115
239
8.72
3
FTU1460A
751
39,461
5
68212
409,272
81,854
217
8.58
4
FTU1317A
826
42,178
8
93329
559,974
69,997
186
7.84
5
FTU0742B
817
40,092
6
24377
146,262
24,377
65
2.60
6
FTU1631A
1003
48,564
14
92387
554,322
39,594
105
5.11
7
FTU0279B
1042
48,021
12
101492
608,952
50,746
135
6.47
8
FTU0640B
979
44,698
13
188760
1,132,560
87,120
231
10.34
9
FTU1317B
862
40,649
7
61224
367,344
52,478
139
5.66
10
FTU1531A
694
36,631
9
110549
663,294
73,699
196
7.17
11
FTU0789A
670
37,794
10
36273
362,730
36,273
96
3.64
12
FTU1484B
817
38,391
3
31023
310,230
103,410
275
10.54
13
FTU0410A
823
41,539
12
40576
405,760
33,813
90
3.73
14
FTU0064B
844
40,815
7
39874
398,740
56,963
151
6.17
Slide 14
Direct comparison of synthesis levels from
full vs. partial genes
MWs
250
150
L: ladder
1: FTU 0504A
100
75
2: FTU 0504Aa
3: FTU 0504Ab
50
4: FTU 0640B
37
5: FTU 0640Ba
25
6: FTU 0640Bb
20
7: FTU 1317A
15
8: FTU 1317Aa
9: FTU 1317Ab
10
Slide 15
MS26.2 Conclusions
• We demonstrated efficient synthesis
of FTU proteins in high throughput
format.
• Large LEE cassettes can be
successfully used in IVT reactions
• Proteins with MW up to 50Kd (~ 500
amino acids in lengths) can be
produced in vitro
Slide 16
MS26.3 : Develop protein
purification protocols
Slide 17
MS26-3. Previous status
Slide 18
Testing NEB IVT kit for crossreactive components in murine
splenocytes
• NEB pure IVT kit contains only essential components
for transcription and translation
• This limits the diversity of E.coli proteins exposed to
T-cells
Slide 19
Reconstituted vs. lysate
IVT systems
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 murine T-cell assay
Slide 20
Determining antigen sensitivity of
our T cell assays, for IVTsynthesized ovalbumin
polypeptide
Slide 21
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 22
Planned Covalent attachment of tag
antibody to beads
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 23
MS26-3. Update
Slide 24
New affinity purification
approach
• Thioredoxin (Trx) tag facilitates proper
protein folding
• Trx tag will be well exposed due to its
large size (11Kda) and folding property
• Protein purification is done using
magnetic beads which are covalently
linked anti-Trx antibody
Slide 25
Choices of antibodies for affinity
purification on beads
1. Anti-his polyclonal (Genentech)
2. Anti-thioredoxin monoclonal
a. AbCam
b. Genescript
3. Anti-thioredoxin polyclonal (Genescript)
Slide 26
Immunoblot analysis of affinity reagents
against recombinant purified Trx protein
AbCam
1
2
3
4
1 2
3
4
Genescript
1
2 3
4
1
2
3 4
1 2
Genetech
3
4
1 2 3
4
R:\GeneVac\FTU\Contract\Proteome\FTU IVT Data\FTU gels\FTU IVT Western\Thio mAb and pAb test 02 on Ecoli and Trx 6-25-08
1: Kaleidoscope ladder, 2: E.coli IVT lysate, 3: Purified recombinant Trx, 4: Chemluminescent
ladder
Slide 27
Conclusions
These results indicate that the
polyclonal and only 1 monoclonal
(mAb A00180) reacts specifically and
efficiently with the thioredoxin in a
western
Slide 28
G-α-Thio
ELISPOT analysis of Thio tagged IVT products
purified on anti-thio, anti-his, or nickel attached
magnetic beads.
Ni
G-α-His
No
Template
No
Template
Ova
FTU901 FTU1695
50% Naïve+
50% DO11.10
Ova
FTU901
FTU1695
LVS Vaccinated
Slide 29
Conclusions
•
•
We observe significant differences among bead
configurations. We are testing the thio G bead, Ni
bead or His G bead bound samples in each row to
assess level of cross-reactivity remaining in
samples after affinity purification of polypeptides
in these 3 different bead-based antigen stimulation
protocols.
the anti-Thio G beads provide the lowest
background, and presumably the highest level of
purification.
Slide 30
Visualizing efficiency of anti-Trx antibody capture of
the Trx-fusion products from IVT lysates
Slide 31
Conclusion
Since the polyclonal antibody is a
less defined sample, and carries
more irrelevant proteins (as
visualized in silver stain) we chose
to proceed with the monoclonal
Genescript Trx antibody.
Slide 32
Pilot Testing of Trx-IVT
samples on NHP IFN-gamma
response assay at UNM
Slide 33
1st NHP ELISpot analysis of cross-reactivity caused by total IVT
lysate proteins vs. IVT lysate proteins after bead purification
FF-LVS antigen
Titration:
Slide 34
Conclusion
• Antigen stimulation using polypeptides
attached to anti-Trx beads significantly
decreases amount of cross-reactivity in
the NHP immune assay
Slide 35
2nd NHP ELISpot analysis of cross-reactivity caused by
total IVT lysate proteins versus NEB pure components
Ova antigen
generated
in E. coli
lysate, and
bound to
anti-trx
beads.
Amount of
Ab on
beads
titrated at
10ul, 20ul,
50ul
FF-LVS antigenTitration:
Blank
Titration
of NEB
pure IVT
mix
Titration
of E.
coli
lysate
“as is”
Slide 36
Third NHP: ELISpot analysis of cross-reactivity
caused by total IVT lysate proteins versus NEB
pure components
E. Coli total IVT lysate
5ul
0.5ul
0.05ul
0.005ul
NEB Pure IVT Mix
FF-LVS
antigen
Titration
Slide 37
Conclusion
The NEB IVT mix displays
significantly less cross-stimulation of
the NHP immune cells as compared to
the full E. coli lysates.
Slide 38
Next steps
•
•
Evaluate IFN-gamma stimulation of
NHP cells by ova and tul4
polypeptides generated with NEB
pure then added “as is” or on antiTrx beads
Test sensitivity of frozen NHP cells
as compared to fresh samples.
Slide 39
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
Inactive
Preparing plan
Inactive
Slide 40
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 41
Previous Status
• Initial qPCR validation studies revealed good results for
FTT901
• LAPT analyses have been performed on the original time
course samples (1,3,5,7, and 24 hours post challenge)
Slide 42
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
Slide 43
Validation of qPCR Primers
(Representative graphs)
FTT0721
FTT0058
Melting Curve
Quantitation
0.001
0.01
0.1
0.001
0.01
0.1
Slide 44
Reconstitution Samples Assessed by qPCR for FTT0721
Melting Curve
Amplification Plot
Genomic DNA Dark Blue
cDNA Light Blue
Negative Pull Down Peaks
Standard Curve
0.01
0.1
Slide 45
Mean Cycle Threshold (Ct) Values for
qPCR Analysis of Reconstitution cDNA Samples
Input RNA
ng/ml
FTT0548 FTT0901 FTT0721
FTT0058
FTT1712c
1000
19.64
18.59
17.86
15.98
11.37
100
24.12
19.96
19.55
19.46
13.09
10
26.34
24.3
23.48
22.97
16.9
1
30.61
28.29
26.7
26.05
20
0.1
ND
30.07
30.01
29.76
23.58
0.01
ND
34.93
32.66
31.68
26.89
0.001
ND
ND
ND
ND
30.09
0
ND
ND
ND
ND
ND
ND = not detected
Slide 46
Conclusions
• We have validated 6 qPCR primer sets with
genomic DNA
• Reconstitution samples (SCHU S4 RNA
diluted into normal mouse lung RNA) have
been assayed by the qPCR system
• With 4 genes we could determine Ct values
down to 0.01 ng/ml SCHU S4.
Slide 47
Upcoming Transcriptome Goals
• Q-PCR validation of the hits
•Design and validate genes for normalization
of qPCR results
•Run qPCR on the original time course
samples
•Time Course Experiment Repeat
•Repeat LAPT of first experiment
•Rat and Mice
•Challenge with 104 SCHU S4 organisms
•Harvest 1,3,5,7 and 24 hours
•Parallel cultures in Chamberlain’s medium
Slide 48
Action Items
•
•
•
•
•
Terry/Rick will analyze mass needed, post purification, in the Elispot assay
and get back to Kathy before ASU scales up the NEB synthesis
Terry will email Kathy and request tul 4 with NEB vs Invitrogen reaction
products.
Terry will email Kathy and request tul 4 product made with NEB vs
Invitrogen reactions.
Terry is preparing SCHU S4 RNA and DNA for ASU
Next ASU technical call is on 8/26/08. Will include the transcriptome
progress and also the past 6-12 months of QA. Marlene will participate on
8/26/08.
Slide 49