Transcript UNM TVDC ASU-_7-31-2007 Slides Minutes_Final

TVDC Progress Report
for ASU
7/31/07
Kathryn F. Sykes and Stephen A. Johnston
Completed Milestones: 25 and 32
Active Milestones: 26, 28, 33, 34, 35
Currently Inactive Milestones: 30, 36-38
CENTER FOR
Slide 1
ASU-TVDC
INNOVATIONS IN MEDICINE
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
further optimized as needed
for purification
in vitro protein yields
are optimized
Purified vs. unpurified samples
are ready for being at NM
CENTER FOR
Slide 2
ASU-TVDC
INNOVATIONS IN MEDICINE
Aim 1
Test ORF synthesis and select expression
constructs
CENTER FOR
Slide 3
ASU-TVDC
INNOVATIONS IN MEDICINE
Aim 1. Previous Status
• His was chosen over biotinylation as a tag for affinity
purification of IVT products.
• Binding efficiency of the tagged products to the Ni-column
varies among different polypeptide samples.
• Two strategies were considered: 1) Maximize opportunities for
tag exposure by adding tags at both ends of the protein; 2) limit
purification to crude removal of IVT components by acetone
precipitation.
CENTER FOR
Slide 4
ASU-TVDC
INNOVATIONS IN MEDICINE
Aim 1. Current Status
•
To evaluate efficiency of the double tag strategy we
designed:
– A simplified IVT cassette. Namely, the sequences for biotin
purification (the TEV and BAP sites) are removed,
– A new 3’ portion of the LEE assembling cassette. Namely, a
C-terminal 6xHis tag is added to the product.
•
To anticipate an approach without affinity purification, we
designed a tag-less cassette:
– A simplified IVT cassette without any extra sequence added to
the protein is designed.
CENTER FOR
Slide 5
ASU-TVDC
INNOVATIONS IN MEDICINE
Aim 1. Current status
The latest set of modular components for IVT LEE assembling
T7
RBS ATG
T7
RBS ATG
T7
RBS ATG
T7
RBS ATG
His
His
ORF
Term
N-term tag
ORF
His
Term
C-term tag
ORF
His
Term
double-tag
Term
w/out-tag
ORF
CENTER FOR
Slide 6
ASU-TVDC
INNOVATIONS IN MEDICINE
Aim 1. Current status
Transcription/translation efficiency of crude vs. perfect PCR products
MW
# Met
ul sample
CPM ul
CPM
CPM Total
CPM #met
ug prot
FTU 728a_a Clone
21,703
4
85
5
11274
191,658
47,915
20.74
FTU 728a_b Clone
24308
4
85
5
12653
215,101
53,775
26.07
FTU 1434c_a Clone
19007
5
85
5
20006
340,102
68,020
25.79
FTU 728a_a mix
21,703
4
85
5
3725
63,325
15,831
6.85
FTU 728a_b mix
24308
4
85
5
13990
237,830
59,458
28.83
FTU 1434c_a mix
19007
5
85
5
23193
394,281
78,856
29.90
CENTER FOR
Slide 7
ASU-TVDC
INNOVATIONS IN MEDICINE
Conclusions
• The developed linear template assembling protocol is ready for
HTP IVT LEE production.
• We developed a modular system allowing quick modification of
the existing IVT cassette.
• Effect of new modifications on IVT yield and purification
efficiency is under evaluation.
• LEE production will start upon completion of the above
evaluation and the ongoing T-cell stimulation experiments.
CENTER FOR
Slide 8
ASU-TVDC
INNOVATIONS IN MEDICINE
Aim 2
Select and test IVT Protocols
CENTER FOR
Slide 9
ASU-TVDC
INNOVATIONS IN MEDICINE
Aim 2. Previous Status
• We have optimized conditions of IVT protein production from
linear PCR generated templates. The optimal reaction include:
– Spike of a feeding solution 30 min after beginning of the reaction
(~100% increase)
– Spike of a linear template 3 hrs after beginning of the reaction
(~20% increase)
– 12 hrs reaction extension (~10% increase)
CENTER FOR
Slide 10
ASU-TVDC
INNOVATIONS IN MEDICINE
Aim 2. Current status
Test of IVT protein production in HTP format (w/out template spike)
MW w/
Prom
# Met
ul sample
ul for
CPM
CPM
CPM Total
CPM #met
ug prot
1
CAlM3
19,500
10
85
5
99733
1,695,461
169,546
63.88
2
FTU 582 B
13,277
2
85
5
9494
161,398
80,699
20.70
3
FTU 887 A
20,851
3
85
5
11895
202,215
67,405
27.16
4
FTU 329 A
14,567
6
85
5
22075
375,275
62,546
17.60
5
FTU 319 A
18,743
5
85
5
23286
395,862
79,172
28.67
6
FTU 1550 A
22,611
9
85
5
35165
597,805
66,423
29.02
7
FTU 1284 A
11,573
4
85
5
30344
515,848
128,962
28.84
8
FTU 1368 B
23019.7
6
85
5
23758
403,886
67,314
29.94
9
FTU 784 A
15,470
3
85
5
29091
494,547
164,849
49.27
10
FTU 741 A
10,693
3
85
5
36536
621,112
207,037
42.78
11
FTU 196 A
24,698
5
85
5
18077
307,309
61,462
29.33
12
FTU 1472 A
13,882
3
85
5
17402
295,834
98,611
26.45
13
FTU 150 A
11,956
3
85
5
19390
329,630
109,877
25.38
CENTER FOR
Slide 11
ASU-TVDC
INNOVATIONS IN MEDICINE
Conclusions
• Applied in HTP format, the developed protocol reliably
generates ~ 20ug of protein per reaction.
• By applying the current reagents and protocols, yield can be
increased by i) additional template spike, ii) prolonged
incubation, iii) increased number of reactions.
CENTER FOR
Slide 12
ASU-TVDC
INNOVATIONS IN MEDICINE
Aim 3
Select and test protein purification protocols
CENTER FOR
Slide 13
ASU-TVDC
INNOVATIONS IN MEDICINE
Aim. 3 Previous Status
• We narrowed the purification process to:
–
–
–
–
–
–
acetone precipitation of the IVT reaction
protein solubility in presence of urea
binding of the de novo synthesized protein to Ni-beads
washing in presence of urea
elution of the bound proteins with imidazole
precipitation of purified protein with acetone
• We found that purification process is associated with
significant loses of the IVT synthesized products
– ~ 50% during acetone precipitation/urea solubilization
– At least 50% during Ni-His purification affinity
– Overall loses between 75% and 100%
CENTER FOR
Slide 14
ASU-TVDC
INNOVATIONS IN MEDICINE
Aim 3. Current Status
IVT samples for evaluation of protein purity on T-cell stimulation
MW
Mets
ul sample
ul for CPM
CPM
CPM Total
Total ug
FTU 1419
1
IVT total
15704
3
450
5
27,335
2,460,150
178.20
2
IVT for Ship
15704
3
100
5
27,335
546,700
39.60
3
8M Urea for Ship
15704
3
100
5
12,142
242,840
17.59
9
Ni Elution
15704
3
100
5
3,726
74,520
7.43
FTU 1695
1
IVT total
14185
5
500
5
27,271
2,727,100
107.06
2
IVT for Ship
14185
5
100
5
27,271
545,420
21.41
3
8M Urea for Ship
14185
5
100
5
17,185
343,700
13.49
9
Ni Elution
14185
5
100
5
10,916
218,320
12.16
GFP
1
IVT total
26000
7
500
5
33,074
3,307,400
169.99
2
IVT for Ship
26000
7
100
5
33,074
661,480
34.00
3
8M Urea for Ship
26000
7
100
5
22,091
441,820
22.71
9
Ni Elution
26000
7
100
5
9,902
198,040
16.40
No Template
1
IVT
100
5
6,195
123,900
2
8M Urea
100
5
1,332
26,640
CENTER FOR
Slide 15
ASU-TVDC
INNOVATIONS IN MEDICINE
Aim 3. Current Status
Additional IVT samples sent to UNM for T-cell stimulation
Protein
MW
ug
1
FTU 1602
15,393
179.33
2
FTU 1696 Aa
21,612
43.80
3
FTU 1696 Ab
26,139
21.14
4
FTU 1696 Ba
23,795
34.63
5
FTU 1696 Bb
22,733
25.64
6
FTU 1712
26,032
91.06
7
FTU 901
18,522
69.98
CENTER FOR
Slide 16
ASU-TVDC
INNOVATIONS IN MEDICINE
Conclusions
•
The current Ni-His tag purification protocol is associated with
significant loses of the in vitro synthesized proteins.
•
The protocol is limited to proteins with unmasked N-terminal
tag. Implementation of C-terminal His tag will expand the list of
nickel-binding samples, but impact on final yields is unclear.
CENTER FOR
Slide 17
ASU-TVDC
INNOVATIONS IN MEDICINE
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
W.T. ORFs
will be finished
In ~1 week
Inactive
Inactive
CENTER FOR
Slide 18
ASU-TVDC
INNOVATIONS IN MEDICINE
Build ORF expression library
corresponding to proteome
CENTER FOR
Slide 19
ASU-TVDC
INNOVATIONS IN MEDICINE
Previous Status
In pilot runs, we were able to generate high quality FTU
natural sequence (WT) ORFs with HTP protocols
CENTER FOR
Slide 20
ASU-TVDC
INNOVATIONS IN MEDICINE
Current Status
• Gene specific primer re-arraying is in progress and should be
completed in a week.
• ORF amplification protocols are in place.
• Templates and primers for promoter and terminator
amplification have been tested and readily available.
• HTP LEE assembling and purification protocols have been
optimized and tested.
• Database management and tracking system is in place.
• We are waiting for the final decision on the IVT purification
format to initiate HTP ORF/LEE/protein production
CENTER FOR
Slide 21
ASU-TVDC
INNOVATIONS IN MEDICINE
MILESTONE 33
Printing and testing
GDP confirmed
Printing arrays
Gray: (sub )milestone title
Red: completed
Green: in progress
GDP Confirmation
Comparisons of substrate
Poly-L Lysine vs Corning Ultragaps
Compare TIGR PFGR Arrays to in house arrays
Testing of linear amplification of procaryotic
Transcripts (LAPT) process and dilution testing of
Schu S4 RNA with and without mouse lung RNA
RNA shipped 1/29/2007
CENTER FOR
Slide 22
ASU-TVDC
INNOVATIONS IN MEDICINE
Genes Identified After Amplification
Amplified 0.01 mg
LAPT-8
Amplified 0.01 mg
LAPT-9
64
11
158
59
0
92
0
1426
Amplified 0.001 mg
LAPT-9
CENTER FOR
Slide 23
ASU-TVDC
INNOVATIONS IN MEDICINE
Genes Identified After Amplification
Amplified 0.01 mg
LAPT-8
Amplified 0.01 mg
LAPT-9
64
25
198
45
0
55
0
1425
Amplified 0.0001 mg
LAPT-9
CENTER FOR
Slide 24
ASU-TVDC
INNOVATIONS IN MEDICINE
Genes Identified After Amplification
Amplified 0.001 mg
LAPT-8
Amplified 0.001 mg
LAPT-9
39
12
48
40
0
60
0
1811
Amplified 0.0001 mg
LAPT-9
CENTER FOR
Slide 25
ASU-TVDC
INNOVATIONS IN MEDICINE
Milestone 33 Summary
• The number of consistently detectable
genes diminishes with dilution
• Amplifications still able to detect 40 genes
at 0.0001 micrograms of spiked sample
CENTER FOR
Slide 26
ASU-TVDC
INNOVATIONS IN MEDICINE
MILESTONE 34
Pilot studies of optimization of RNA
isolation and hybridization conditions
Gray: (sub )milestone title
Red: completed
Green: in progress
RNA Isolation (UNM
Hybridization Conditions
Initial testing of heavily infected lungs
Perform CFU analyses and compare with purified RNA
Testing Maui Hybridization chamber
Amplification testing of Schu S4 RNA
with and without mouse lung RNA
RNA isolated from infected lungs received from UNM
CENTER FOR
Slide 27
ASU-TVDC
INNOVATIONS IN MEDICINE
Milestone 34 Summary
• Clonetech changes SuperSmart cDNA
Synthesis kit
– Stops providing “Powerscript RT”
• Used recommended replacement
– Amplifications failed
CENTER FOR
Slide 28
ASU-TVDC
INNOVATIONS IN MEDICINE
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
CENTER FOR
Slide 29
ASU-TVDC
INNOVATIONS IN MEDICINE
Milestone 35 Summary
• Received dose response samples from
UNM
• Performed RNAeasy purification
– Purified individual samples
– 3 mice per group
– 103, 104, 105, 106, 107 FTU/Mouse
• Made Pool of each dose
• Amplification Failed
CENTER FOR
Slide 30
ASU-TVDC
INNOVATIONS IN MEDICINE
Upcoming Monthly Transcriptome Goals
• Amplification samples to be retested on TIGR and ASU
arrays
– 10 mg – high intensity signal across most genes
– Use ~10-20 genes from proteome amplification for array QC
– Confirm with Agilent Arrays or Samples from Karl Klose
• QC testing of new MMLV RT for cDNA synthesis and PCR
reaction.
• Begin testing samples of animals challenged with varying
doses of SCHU S4 to determine level of detection.
CENTER FOR
Slide 31
ASU-TVDC
INNOVATIONS IN MEDICINE
Action Items: 7/31/07
• Action: Alex: Is there an explanatory difference for FTU 728a_a PCR
outlier results on slide 7?
• Action: Alex: - the proteomics team could put urea into the IVT
product tube, add magnetic beads and pull product out. Acetone is
good for precipitations but precipitation may make it harder to
resuspend the IVT product for the next step.
• Action: Mitch- ASU has ordered enzymes with appropriate tailing
activity. Making own in house LAPT kit and not be dependent on
Clontech kits for the future. Will be testing samples from UNM in
near future.
CENTER FOR
Slide 32
ASU-TVDC
INNOVATIONS IN MEDICINE