ASU TVDC Progress Report
4/29/08
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
ML26-2 – completed
ML26-3 – pending
a. Construction of a thioredoxin fusion plasmid for
protocol testing.
b. Evaluation of preclearing Ni beads before use in
purification
c. Evaluation of co-incubating Ni beads during IVT
reactions
Slide 3
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
• Decision was made to evaluate methods to
improve folding or alternative methods of
purification that are folding-independent.
Slide 4
ML26-3. Update
Construction of a thioredoxin
fusion plasmid for use as LEE PCR
template and for recombinant
protein production during testing.
Slide 5
Thio-fusion LEE
ORF
T7-Pro
T7-Pro
Thio.
Thio.
T7-Term
6-His
6-His
ORF
T7-Term
Slide 6
Thio-fusion plasmid constructs
• Design primers to use for PCR
amplification of FTU ORF from perfect
clone.
• Cloning of FTU ORF into pET32b
Slide 7
Thio fusion cloning into pET32b
1
1
2
6
2
3
4
3
4 5
6 7
7
8
5
1
8
2
3
4
9
9
Double digestion of FTU ORFs
1-9: Ova, FTU 721A, FTU 721B, FTU
721C, FTU 901, FTU 1695, FTU 1696 Aa,
FTU 1696 Ba, and FTU1712
pET32b double digestion
1: EcoR1 digestion
2: XHoI digestion
3: EcoR1 and XHoI digestion
4: Uncut pET32b
Slide 8
ML26-3. Update
Evaluation of pre-clearing beads
before use in purification
and Evaluation of co-incubating
beads during IVT
Slide 9
IVT “as is” and standard purification
from Ni magnetic beads
Coomassie
Autoradiograph
1
1 2
3
4
5
6 7
1,2. Ovalbumin
8
2
3 4 5
6
7 8 9 10 11 12 13 14 15 16 17 18
9 10 11 12 13 14 15 16 17 18
11,12. FTU 1695
3,4.
FTU 721A
13,14. FTU 1696Aa
5,6.
FTU 721B
15,16. FTU 1696Ba
7,8
FTU 721C
17,18. FTU 1712
Odd numbers: IVT
Even numbers: Elution
9,10. FTU 901
Slide 10
Optimizing amount of Ni-magnetic bead to use
for purification
1 2 3 4 5 6 7 8 9 10
1
SDS gel
2
3 4
5
6
7
8 9 10
autoradiograph
1: IVT, 2: blank
Odd numbers (except 1): Supernatant
3,4: 5ul
Even numbers ( except 2): Elution
5,6: 10ul
Sample is the mixture of: Ova, FTU 721A , FTU 721C,
7,8: 15ul
FTU 1695, and FTU 1696Aa
9,10: 20ul
Slide 11
LEE construction for Rabit Retic
IVT
1
2
3
4
5
6
7
8
9 10
1.
Ovalbumin
2.
FTU 721A
3.
FTU 721B
4.
FTU 721C
5.
FTU 1696Aa
6.
FTU 1696Ba
7.
Calm3 (Not
Used)
8.
FTU 901
9.
FTU 1712
10. FTU 1695
Slide 12
Comparison of total yields from E.coli vs. Rabbit retic IVT
E.coli lysate
(total ug)
Rabbit Reticulocytes (total ug)
Ova
68.04
0.003
FTU 721A
28.72
0.002
FTU 721B
18.69
0.003
FTU 721C
6.51
0.003
FTU 901
27.28
0.004
FTU 1695
17.46
0.005
FTU 1696 Aa
27.67
0.003
FTU 1696 Ba
123.44
0.006
3.43
0.007
Template
FTU 1712
Calculation of based on 100ul reaction in E.coli, and 50ul in RR IVT
Slide 13
Effect of E. coli lysate preclearing on total IVT yield
Total protein (ug)
20
18
Ova
16
FTU 1695
14
12
10
8
6
4
2
0
0
25
50
100
Amount of beads (ul)
Slide 14
Effect of pre-clearing E.coli lysate on
yield of polypeptide binding to Ni-beads
Bead
volume:
Ova
FTU
1695
Without preclearing
IVT
Sup
(ug)
(ug)
Bound
to
bead
25 ul
IVT
Sup
(ug)
(ug)
17.04 11.40 5.63 15.5
8.73 1.12 7.61 6.9
7
Bound
to
bead
50 ul
2.5 4.4
IVT
Sup
(ug)
Bound
to
bead
(ug)
(ug)
Bound
to
bead
6.9
10.4
9.8
4.1
5.7
3
6.6
3.7
1.3
2.4
IVT
Sup
(ug)
8.5 17.3
9.6
100 ul
Slide 15
Effect of urea vs. co-incubation of beads
during IVT reaction on efficiency of
polypeptide binding to magnetic Ni beads
ova
FTU 721A FTU 721B FTU 721C
FTU 901
FTU 1695 FTU1695Aa FTU1696Ba FTU 1712
GFP-trix
1 23 1 2 312 3 1 2 31 2 3 1 231 23 12 3 1 2 31 23
1: 10ul IVT
2: 20ul supernatant of 6M urea denatured protein bound on Ni magnetic beads
3: 10ul supernatant from protein purification from Ni magnetic beads incubated
during IVT reaction
(All have been normalized to 10% of total volume)
Slide 16
Effect of blocking Ni beads with
fetal bovine sera prior to
polypeptide binding
Without blocking
(8M urea)
FBS blocking
Ova
81%
61%
FTU 1695
80%
65%
Data represents estimated percentage of proteins bound to the beads
Slide 17
Samples for UNM Elispot-Trial 1
1.
2.
3.
4.
5.
6.
FTU protein with E.coli IVT “as is”.
Traditional affinity purification using Ni magnetic beadsElution fraction.
Incubation of Ni beads during IVT reaction. Beads were
washed. After washing, the beads were resuspended in PBS.
Denature IVT proteins with 6M urea prior to binding to the Ni
magnetic beads. After washing, the beads were
resuspended in PBS.
Substitute Ni-magnetic beads with protein G-beads. IVT
proteins bound to the beads via anti-His antibody. Beads
were washed. After washing, the beads were resuspended in
PBS.
FTU protein with Rabbit Reticulocyte IVT “as is”.
Slide 18
ELISpot of splenocytes from Ova mice
Ovalbumin
Ovalbumin(1/10)
FTU 721A
FTU 721B
Headings for FTU rows
Column 7: IVT “as is”
Column 8: elution IVT fraction after
dialysis
Column 9: non-denatured IVT proteins
bound to beads
Column 10: 6M urea denatured
proteins bound to beads
Column 11:IVT proteins bound to
protein G
Column 12:Rabbit retic IVT “as is”
FTU 721C
FTU 901
FTU 1695
Control row:
FTU 1696Aa
FTU 1712
FTU 1696Ba
Control row:
Control row:
No Ag
OVA
peptide
Sigma
OVA
protein
Blank
protein G
beads
Blank Ni
beads
IVT no
template
(C12)
Slide 19
ELISpot of splenocytes from vaccinated mice
Ovalbumin
Ovalbumin(1/10)
Headings for FTU rows
Column 7: IVT “as is”
Column 8: elution IVT fraction after
dialysis
Column 9: non-denatured IVT proteins
bound to beads
Column 10: 6M urea denatured
proteins bound to beads
Column 11:IVT proteins bound to
protein G
Column 12:Rabbit retic IVT “as is”
FTU 721A
FTU 721B
FTU 721C
FTU 901
FTU 1695
Control row:
FTU 1696Aa
FTU 1712
FTU 1696Ba
Control row:
Control row:
No Ag
OVA
peptide
Sigma
OVA
protein
Blank
protein G
beads
Blank Ni
beads
IVT no
template
(C12)
Slide 20
Samples for UNM Elispot-Trial 2
1. Clearing of Ni binding proteins from
E.coli lysate prior to IVT reaction
2. Blocking Ni magnetic beads with Fetal
Bovine Sera prior to binding of FTU
proteins to the beads
Slide 21
O
V
A
L
V
S
Sample rows
control rows
ivt no
template
25 µL Ni
beads
Sigma
OVA
protein
OVA
25 FTU 1695
µL Ni
25 µL Ni
beads
beads
Sigma
OVA
protein
Sigma
OVA
protein
ivt no
template
50 µL Ni
beads
OVA
peptide
ivt no
OVA
50 FTU 1695 template
OVA
FTU 1695
µL Ni
50 µL Ni 100 µL Ni 100 µL Ni 100 µL Ni
beads
beads
beads
beads
beads
OVA
peptide
OVA
peptide
No Ag
No Ag
ivt no
template
BSA
OVA
BSA
FTU 1695
BSA
No Ag
Slide 22
Conclusions
• Adding the Ni beads during the IVT
reactions significantly improves
polypeptide “capture”, thereby
improving yields
• Pre-clearing the IVT lysates with beads
prior to IVT, significantly reduces nonspecific T cell reactivity
• …Without reducing specific reactivity
Slide 23
Go/No Go decisions update
• We will prepare all antigen samples as
described above- E. coli purified and
Rabbit Retic unpurified -to UNM.
• Based on antigen specfic and nonspecific reactivities in UNM ELISpot
assays we will select a method for
antigen production.
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
On-hold
Inactive
Slide 25
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 and Time Course of Infection
QPCR Primers in Design
Slide 26
Previous Status
•Initial studies performed with two biological experiments
focusing on dose-response of challenge
•Initial time was 4 hours post-challenge
•RNA from each experiment was amplified twice and the
most consistently detected genes were combined from the
key doses (103-107)
•141 genes listed
Slide 27
Time Course of Gene Expression Changes
Decreasing
Signal Intensity
Increasing
T0
T1
T3
T5
T7
T24
T1
T3
T5
T7
T24
T0
Slide 28
Overlap of Increasing and Decreasing with
the Dose Response Picks
Decreasing
Increasing
6
94
91
0
0
2
139
141 – 4 Hr Dose Response
Slide 29
Signal Intensity
Expression Patterns of
Putative Vaccine Candidates
T0
T1
T3
T5
T7
T24
Slide 30
Expression Levels of Two Genes Identified
By Multiple Assays
FTT1616
FTT0031
Cysteinyl-tRNA synthetase
NADH dehydrogenase I, A subunit
Slide 31
Conclusions
• Initial time course analysis reveals genes of increasing
and decreasing expression during the first 24 hours of
infection
• There is minimal overlap with the 4 hour gene list
identified from the dose response experiments
Slide 32
Upcoming Transcriptome Goals
• Q-PCR validation of the hits
• Primers being designed
• groEL / icglC / dnaK / tul4 / katG
• Time Course Experiment
• Repeat amplification of time course samples
• Parallel cultures in Chamberlain’s medium
Slide 33
Action Items
• Kathy: titrate above 20ul of Ni beads to
possibly improve yield of purified IVT
products
• Mitch/Rick/Stephen: At the 5/16/08 ASU
site visit, we need to plan the other gene
expression experiments to accomplish in
the remaining time of the ASU subcontract
perhaps using other tissues, beside the
lungs, comparing rat vs mouse as
different species, etc. Microarrays are
running well, so now need a pipeline in
place.
Slide 34
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 and Time Course of Infection
QPCR Primers in Design
Slide 35
Previous Status
•Initial studies performed with two biological experiments
focusing on dose-response of challenge
•Initial time was 4 hours post-challenge
•RNA from each experiment was amplified twice and the
most consistently detected genes were combined from the
key doses (103-107)
•141 genes listed
Slide 36
Time Course of Gene Expression Changes
Decreasing
Signal Intensity
Increasing
T0
T1
T3
T5
T7
T24
T1
T3
T5
T7
T24
T0
Slide 37
Overlap of Increasing and Decreasing with
the Dose Response Picks
Decreasing
Increasing
6
94
91
0
0
2
139
141 – 4 Hr Dose Response
Slide 38
Signal Intensity
Expression Patterns of
Putative Vaccine Candidates
T0
T1
T3
T5
T7
T24
Slide 39
Expression Levels of Two Genes Identified
By Multiple Assays
FTT1616
FTT0031
Cysteinyl-tRNA synthetase
NADH dehydrogenase I, A subunit
Slide 40
Conclusions
• Initial time course analysis reveals genes of increasing
and decreasing expression during the first 24 hours of
infection
• There is minimal overlap with the 4 hour gene list
identified from the dose response experiments
Slide 41
Upcoming Transcriptome Goals
• Q-PCR validation of the hits
• Primers being designed
• groEL / iglC / dnaK / tul4 / katG
• Time Course Experiment
• Repeat amplification of time course samples
• Parallel cultures in Chamberlain’s medium
Slide 42
Action Items
• Kathy: titrate above 20ul of Ni beads to
possibly improve yield of purified IVT
products
• Mitch/Rick/Stephen: At the 5/16/08 ASU
site visit, we need to plan the other gene
expression experiments to accomplish in
the remaining time of the ASU subcontract
perhaps using other tissues, beside the
lungs, comparing rat vs mouse as
different species, etc. Microarrays are
running well, so now need a pipeline in
place.
Slide 43