ASU TVDC Progress Report
02/26/08
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 to His/Ni
purification is being
investigated
Slide 2
Milestone 26
ML26-1 – completed
ML26-2 – completed
ML26-3 – Will close 03/01/08
a. Evaluate feasibility of the IVT depletion approachongoing.
b. Evaluate feasibility of switching from an E. coli to
a wheat germ or rabbit reticulocyte IVT systemscompleted.
c. Generate IVT polypeptides and related reagents
for optimizing UNM’s antigen-screening T cell
assay-ongoing.
Slide 3
ML26-3. Previous Status
a. A set of size-fractionated IVT samples were generated at
ASU and tested at UNM for the presence/absence of the
cross-reacting E. coli antigen in the IVT lysate. Filter units
with 100 kDa size cutoffs deplete the cross-reactive
component.
b. Prolonged incubation and shaking does not disrupt large
complexes (macrocomplexes) in lysate.
c. A Freeze/ thaw cycle does destabilize macrocomplexes.
d. Filtration enriches polypeptide in lysate.
e. Nonetheless, filtration traps more than 50% of the
polypeptide products, even with the 100 kDa unit
Slide 4
ML26-3. Previous Status
f.
Filtration removes ~90% of total E. coli proteins in lysate.
g.
Dilution of lysate sample or addition of detergent has no
significant effect on protein depletion.
A 40% acetone “cut precipitates most of the large E.coli
proteins and/or complexes,
h.
i.
Slide 5
Yet maintains 3 smaller proteins, <40kDa proteins, in the
supernatant.
ML26-3. Previous Status
Slide 6
j.
Acetone cut enables very high efficiency purification of
affinity-tagged product from lysate.
k.
Same problem of significant sample losses (50%) is
associated with purification from acetone depleted IVT
reactions.
l.
This is no more or less than losses also observed with
purified recombinant proteins.
ML26-3. Update
Slide 7
Temperature dependent acetone
precipitation of 35S-IVT products
1
2
3 4
5 6 7
8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
FTU 721A
Slide 8
FTU 1696Aa
FTU 1695
1.
RmTemp pellet
9.
Complete IVT sample
18.
RmTemp pellet
2.
RmTemp Super
RmTemp Super
4oC Pellet
RmTemp pellet
RmTemp Super
4oC Pellets
19.
3.
10.
11.
12.
20.
4oC Pellets
4.
4oC Super
13.
4oC Sup.
21.
4oC Sup.
5.
1:10 dil. RmTemp pellet
22.
1:10 dil. RmTemp pellet
6.
1:10 dil. RmTemp pellet 14.
1:10 dil RmTemp Super 15.
23.
1:10 dil RmTemp Super
7.
1:10 dil. 4oC pellet
16.
1:10 dil. 4oC pellet
24.
1:10 dil. 4oC pellets
8.
1:10 dil. 4oC Super
17.
1:10 dil. 4oC Super
25.
1:10 dil. 4oC Sup.
1:10 dil RmTemp Super
Effect of time on separation of 35Sproducts by 4oC acetone
1
2
3 4
5 6 7
8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
FTU 721A
Slide 9
FTU 1696Aa
FTU 1695
1.
15 min. pellets
9.
Complete IVT sample
18.
15 min. pellets
2.
15 min. Sup.
10.
15 min. pellets
19.
15 min. Sup.
3.
30 min. pellets
11.
15 min. Sup.
20.
30 min. pellets
4.
30 min. Sup.
12.
30 min. pellets
21.
30 min. Sup.
5.
45 mi. pellets
13.
30 min. Sup.
22.
45 min. pellets
6.
45 min. Sup.
14.
45 min. pellets
23.
45 min. Sup.
7.
60 min. pellets
15.
45 min. Sup.
24.
60 min. pellets
8.
60 min. Sup.
16.
60 min. pellets
25.
60 m in. Sup.
17.
60 min. Sup.
Effect of time on precipitation of E. coli lysates
by 4oC acetone
250
150
Same
legend
as slide
#8
1
2
3 4
5 6 7
8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
100
75
50
Tempera
ture
37
25
20
15
10
FTU 721A
1
2
3 4
5 6 7
FTU 1695
FTU 1696Aa
8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
Same
legend
as slide
#9
Incubation
time
FTU 721A
Slide 10
FTU 1695
FTU 1696Aa
Conclusions
• A 4oC acetone precipitation step separates E. coli
proteins from de novo polypeptides
• A 1:10 dilution of lysate before acetone ppt allows
more of both E. coli proteins and de novo products to
remain in the super, so does not facilitate separation.
•A 15-30 min incubation with 4oC acetone is sufficient
to precipitate E. coli proteins and allows polypeptides
to remain in super.
Slide 11
IVT reaction incubation time
Testing polypeptide
product stability:
6.0
5.0
•Samples were collected
at indicated hours
4.0
3.5
3.0
2.0
0.5
1.0
FTU 1695
FTU 721a
•Reaction was stopped
after 6 hours of
incubation
Results:
IVT products from of partial ORF template (FTU721A) produced incomplete translation products
as compared to the full length template (FTU 1695)
Slide 12
Total IVT polypeptide Production vs.
Reaction Time
20.00
18.00
ug total protein
16.00
14.00
12.00
10.00
8.00
6.00
4.00
2.00
0.00
0
1
2
3
4
5
6
Hours of incubation
FTU 721A
Slide 13
FTU 1695
7
Testing IVT protein dilution for acetone
precipitation
lysate
P
FTU 721a
1:1
1:3
S
P
P: pellet
S: Supernatant
Dots show positions of
molecular marker
Slide 14
S
P
FTU 1695
1:7
S
P
S
lysate
1:1
P
P
S
1:3
S
P
1:7
S
P S
Conclusions
1. Dilutions do not provide a significant
difference in acetone precipitation of IVT
proteins
2. Precipitation of fragment FTU 721A takes
place at even lower percentage of acetone
in compared to FTU 1695; a stable, soluble,
and full length protein
3. Cannot use acetone for precipitation in high
through put aspect unless all proteins are
stable and folding properly
Slide 15
His-tag detection
1
2
3
4
5
6
7
8
2
3
4
5
6
7
8
9
Western Blot using anti-His antibody
Slide 16
Molecular marker
2.
IVT FTU 721A
3.
IVT FTU1696Aa
4.
IVT FTU 1695
5.
0.1 ug
6.
0.5 ug
7.
1.0 ug
8.
3.0 ug
9.
5.0 ug
9
His-tag stain
1
1.
Pre-purified FTU 1695
Conclusions
1. We believe the at the His-tag on IVT
polypeptide is buried, and the products
improperly folded. Therefore, the protein
does not efficiently bind to either the antiHis antibody or Nickel in the staining kits.
2. This result explains low yields following all
affinity purification schemes.
3. We conclude that we must re-engineer the
template to facilitate proper protein folding
during in vitro translation.
Slide 17
A ThioFusion based expression
System
• The small 11.7 kDa thioredoxin protein
can be massively over-expressed in E.
coli and remain soluble.
• When fused N-terminally to other
proteins, trx can increase translation
efficiency and solubility
Slide 18
New constructs in progress
ORF
T7-Pro
T7-Pro
Thio.
Thio.
T7-Term
6-His
6-His
ORF
T7-Term
•A new LEE construct has been designed to express
antigens with a thioredoxin (Thio) peptide fusion.
•The presence of this new tag is intended to aid proper
folding of in vitro translated proteins
• IVT rxns will be carried out with these LEEs.
Polypeptide yields, stability, and His-binding will be
evaluated.
Slide 19
LEE construction using p32b
promoter
Amplification of promoter
from p32b plasmid
containing Thioredoxin
fusion tag
LEE constructs in progress
Slide 20
Testing efficiency of E.coli lysate protein depletion with
higher MW-cutoff membrane filter
R
1
2
3
F
R
4
5
F
6
Coomassie
Slide 21
R
F
R
F
7
8
9
10
1.
Molecular marker
2.
IVT rxn mixture 1/10 dil
3.
Retentate from 100kda,1/10
dil.
4.
Filtrate from 100kda,1/10 dil.
5.
Retentate from 300kda, 1/10
dil.
6.
Filtrate from 300 kda, 1/10 dil.
7.
Retentate from 100 kda, no dil
8.
Filtrate from 100 kda, no dil
9.
Retentate from 300 kda, no dil
10.
Filtrate from 300 kda, no dil
Conclusions
• The higher MW cut-off filter unit (300kDa) does not appear to
change total protein separation when the lysates are used neat.
• However, when the lysates are diluted 10-fold, most of the E.
coli proteins pass through the filter, as would be expected.
• We believe that the undiluted samples simply clog filter and no
size separation occurs.
• If our supposition is right, that the cross-reacting proein is very
large, than it may be retained by 300kDa filtration.
Slide 22
ML26-3. Next steps
• Test new 300kDa MW filtrate samples in T cell assays
(sent to UNM last week).
• Determine whether we can isolate the cross-reacting
E.coli antigen into either the pellet or super at some
acetone concentration. We set up a series of acetone
precipitations of IVT lysates. We provided both pellet
and super to UNM for testing in T cells assays. If
successfully isolated, we will aim to direct
polypeptides to the alternative fraction.
• use Thiofusion constructs in IVT reactions and
evaluate efficiency of an affinity purification of
products.
Slide 23
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 24
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
Spearman Correlations of Microarray Data
Between Doses – LAPT 22
102
103
104
105
106
Between Replicates Samples
107
101 0.685
102
103
104
105
106
Slide 26
103
104
105
106
107
103 0.765
104
0.458
105
0.406
106
0.552
107
0.777
0.548
0.746
0.769
0.428
0.168
Comparisons of Genes Amplified from
LAPT- 20
103
104
LAPT - 22
247
181
105
215
Slide 27
252
317
286
250
181
212
Comparisons of Genes Amplified from
LAPT-20
LAPT-22
103
104
103
104
78
63
103
59
129
293
28
Slide 28
148
194
68
112
94
109
101
105
105
Comparisons of Genes Amplified from
LAPT-22
53
Slide 29
LAPT-20
141
152
Raw Signal vs Genomic Normalization
GN
103
103
104
Raw
Slide 30
Microarray Histograms of Replicate Amplifications
LAPT-22
Challenge
dose
Slide 31
102
103
104
105
LAPT-20
106
107
101
102
103
104
105
106
107
Top Gene Hits
Gene Identifier
FTT0050
FTT1443c
FTT0510
FTT1592c
FTT0238
FTT0386
FTT0590
FTT0552
FTT0675
FTT0243
FTT0075
FTT0325
FTT0511
FTT1463c
FTT0680c
FTT0610
FTT1176c
FTT0676
FTT1027c
FTT0117
FTT0699
FTT0919
FTT0557
FTT0163c
FTT0595c
FTT1292c
FTT0687c
FTT0549
FTT0199
FTT0519
FTT1539c
FTT0034
Slide 32
Intensity
65,535
58,553
57,931
51,120
48,645
44,879
44,519
43,783
41,292
36,396
35,939
33,824
32,883
32,368
30,423
29,431
28,535
27,672
27,254
26,313
25,608
25,133
24,334
24,200
23,845
23,447
23,445
22,435
22,263
22,144
20,724
20,206
Name
translation initiation factor IF-2
ATPase, AAA family
DNA gyrase subunit B
Chitinase, fragment
Shikimate 5-dehydrogenase
Nicotinamide-nucleotide adenylyltransferase
Ribonuclease H
aldehyde dehydrogenase
50S ribosomal protein L25
hypothetical membrane protein
succinate dehydrogenase iron-sulfur protein
50S ribosomal protein L3
Pyridoxine/pyridoxal 5-phosphate biosynthesis protein
galactosyl transferase
Peptidyl-tRNA hydrolase
DNA/RNA endonuclease family protein
Type I restriction enzyme subunit R, pseudogene
conserved hypothetical membrane protein
3-deoxy-D-manno-octulosonate 8-phosphate phosphatase
thymidylate kinase
polyribonucleotide nucleotidyltransferase
hypothetical protein
AhpC/TSA family protein
Topoisomerase IV, subunit B
Rubredoxin
conserved hypothetical protein
ATP-dependent protease, ATP-binding subunit
D-alanyl-D-alanine carboxypeptidase
hypothetical protein
Nif3 family protein
conserved hypothetical protein
NADH dehydrogenase I, D subunit
Conclusions
• High dose inhibition observed in previous
amplification did not repeat
• Highest correlations between samples are in
challenge doses from 103-106
• Genomic normalization process may allow for
detection of low-level expressing genes and for
better multi-slide to slide comparisons.
Slide 33
Upcoming Transcriptome Goals
• Q-PCR validation of the hits
• Establish next experimental parameters for MS35
•Time Course Experiment
•Challenge with 104 SCHU S4 organisms
•Harvest 1,3,5,7 and 24 hours
•Parallel cultures in Chamberlain’s medium
Slide 34
Action Items
• Alex and Kathy will develop a budget for rabbit
reticuloycyte lysate while testing thioredoxin for folding
protein fragments in parallel.
• Kathy and Alex need to define “go/no go” decisions for
IVT protein fragment folding approach.
• Barbara will send ASU slides in March, at least 24 hrs
prior to the ASU tech call
• Rick will give ChiChi (At Sandia) Mitch’s email address
to share the metabolic pathway software with ASU.
• Rick will ask Terry the status of the 500ug of SCHU S4
DNA for ASU and the next round of infected lung RNA
samples for ASU (single dose and kinetics)(2/28 status: both
SCHU S4 DNA and mouse kinetic lung RNAs are prepared, sterile, out of the UNM BSL3 and will
be sent to ASU during week of 3/3)
Slide 35