F. t. antigen library production,
purification and pooling
strategies for T cell screen
Evolution of IVT DNA template
▼ Linear fragment replaces plasmid.
• Similar efficiency in standard IVT rxns
▼ 1st generation lnear cassette- MinT7:BAP:UB:His:TEV:ORF:term
• Large size, low expression
▼ 2nd generation cassette- without UB
• Smaller size, higher expression, inefficient biotinylation
▼ 3rd generation cassette- EnhT7, w/o BAP or TEV sites
• Minimized size, optimal expression
▼ Final cassette- EnhT7:His:ORF:His:Term
• Set of modular formats selected for optimal expression and
purification
Effect of His-tag position
TCA Analysis Step
N-tag
C-tag
CPM
prot (ug)
IVT
+
-
1095174
38.2
IVT/6M Guanidine HCL
+
-
828206
28.9
Flow Through
+
-
58344
2.0
Wash
+
-
9048
0.3
Beads Post Wash
+
-
24120
0.8
Elution
+
-
18648
0.7
Beads Post Elution
+
-
5610
0.2
Effect of His-tag position
TCA Analysis Step
N-tag
C-tag
CPM
Protein yield (ug)
IVT
-
+
1078565
37.0
IVT/6M Guanidine HCL
-
+
935476
32.0
Flow Through
-
+
58084
2.0
Wash
-
+
4160
0.1
Beads Post Wash
-
+
68500
2.3
Elution
-
+
8631
0.3
Beads Post Elution
-
+
12620
0.4
IVT
+
+
880396
33.5
IVT/6M Guanidine HCL
+
+
736814
28.0
Flow Through
+
+
62166
2.4
Wash
+
+
5044
0.2
Beads Post Wash
+
+
7240
0.3
Elution
+
+
12579
0.5
Beads Post Elution
+
+
30010
1.1
Current status of HTP plan
• All protocols have been validated and protein
production/purifcation ready
– Single His-tag cassette at C-term
– single IVT reaction with a single feed and without
template spike
– 35S-based protein quantification and detection
• Quality control
– Template (UV quantification, e-gel)
– Product (CPM, e-PAGE , autoradiography)
• Readied modifications if needed
– Switch to N-term or no tag cassette
– Additional template spike, extended incubation
(~10-20% increase in yield)
– +/- purification
IVT protein production is HTP
MW w/
Prom
# Met
CPM
CPM Total
ug prot
1
CalM3
19,500
10
99733
1,695,461
63.88
2
FTU 582 B
13,277
2
9494
161,398
20.70
3
FTU 887 A
20,851
3
11895
202,215
27.16
4
FTU 329 A
14,567
6
22075
375,275
17.60
5
FTU 319 A
18,743
5
23286
395,862
28.67
6
FTU 1550 A
22,611
9
35165
597,805
29.02
7
FTU 1284 A
11,573
4
30344
515,848
28.84
8
FTU 1368 B
23019.7
6
23758
403,886
29.94
9
FTU 784 A
15,470
3
29091
494,547
49.27
10
FTU 741 A
10,693
3
36536
621,112
42.78
11
FTU 196 A
24,698
5
18077
307,309
29.33
12
FTU 1472 A
13,882
3
17402
295,834
26.45
13
FTU 150 A
11,956
3
19390
329,630
25.38
Accuracy of protein
calculation/QC
0.8
0.7
0.6
y = 0.0049x - 0.0011
R2 = 0.9966
0.5
0.4
0.3
0.2
0.1
0
-0.1
0
20
40
60
80
100
Concentration (ug/ml)
S35 based
(ug)
Stain based
(ug)
FTU901
1.24
1.06
FTU1419
0.88
.097
120
140
160
Purity options
• Total IVT reaction
– Mix of all rxn components
– Free 35S label
• Acetone precipitate
- No solubility issues
– Mixture of proteins
- Removal of free label and
other rxn components
– Solubility issues
- Easy storage/shipping
• Purified protein
– Some sample loss
– Variable solubility
- Improved readouts?
Polypeptide purification
approaches
• Removal of non-IVT proteins
– Ammonium sulfate fractionation.
• Tested: Not useful due to the lack of selectivity.
– E. coli protein depletion.
• Not tested: Not attractive due to high costs of resin.
• Isolation of synthesized polypeptides
– Biotin/avidin
• Tested: Not practical due to low biotinylation efficiency.
– His-tag/Ni-Ag
• Tested: Feasible, though yields poor
– His-tag/Ni-Fe
• Tested: feasible, yields significantly improved
•
•
His-tag purification
development
•
•
•
•
Buffers
– 20mM Tris-HCl, pH8.0
– 20mM Tris-phosphate, pH8.0
– 10mM Phosphate, pH7.5
Folding
– Native
– 2M urea
– 8M urea
– 4M guanidine
– 8M guanidine
Resin/beads
– Ni-NTA Qiagen
– Ni-Hitrap Amersham
– Co-Dyna beads, Invitrogen
– Ni-Fe styrene beads
Format
– Batched (tube)
– Column
Wash
– Binding buffer with 20mM imidazole
Elution
– 0.5M EDTA, pH8.0
– HCl, pH4.0
– 0.5M imidazole
– 1.0M imidazole
IVT fractionation with (NH4)2SO4
0%
5 10 20
25% 50% 75% 90%
S
P S P S P S P
0%
5 10 20
25%
S
P
His-tag purification
T W E T W E T W E
T W E T W E
Legend:
T=Total
W=Wash
E=Eluent
Coomassie stained PAGE
Protein
FTU 1419
His tag
purification
efficiency
on Ni+
sepharose
beads
FTU 1695
GFP
Fraction
IVT for Ni
Exp 1
prot (ug)
Exp 2
(%)
prot (ug)
(%)
135.25
100.0%
152.62
100.0%
8 M Urea for Ni
68.49
50.6%
56.56
37.1%
Flow Through
61.15
45.2%
54.99
36.0%
Wash 1
1.52
1.1%
1.43
0.9%
Wash 2
0.69
0.5%
0.44
0.3%
Elution
7.37
5.5%
2.82
1.8%
IVT for Ni
97.15
100.0%
82.22
100.0%
8 M Urea for Ni
58.16
59.9%
33.05
40.2%
Flow Through
23.80
24.5%
23.22
28.2%
Wash 1
4.03
4.1%
2.46
3.0%
Wash 2
2.64
2.7%
1.49
1.8%
Elution
12.96
13.3%
5.52
6.7%
IVT for Ni
84.16
100.0%
64.56
100.0%
8 M Urea for Ni
53.40
63.5%
31.39
48.6%
Flow Through
32.42
38.5%
22.78
35.3%
Wash 1
2.56
3.0%
1.51
2.3%
Wash 2
2.15
2.6%
0.82
1.3%
Elution
8.40
10.0%
5.22
8.1%
Ni-Fe bead-based purification
MW
CPM Total
ug prot
% yield
CalM
1 IVT
19469
615042
17.4
100.0
110520
3.1
18.0
3 Wash1
10380
0.3
1.7
4 Wash2
3800
0.1
0.6
5 Elution
334860
9.5
54.4
129042
8.4
100.0
49380
3.2
38.2
3 Wash1
4420
0.3
3.4
4 Wash2
680
0.0
0.5
5 Elution
32040
2.1
24.8
2 Flow through
GFP
1 IVT
2 Flow through
26995
MW
CPM Ttl
ug prot
% yield
Ni+ Fe #1
IVT
15738
131200
6.5
99.9
39400
2.0
30.0
Wash 1
7120
0.4
5.4
Wash 2
4000
0.2
3.0
Elution
30200
1.5
23.0
131200
6.5
99.9
40880
2.0
31.1
Wash 1
6640
0.3
5.1
Wash 2
2400
0.1
1.8
Elution
43560
2.2
33.2
131200
6.5
99.9
45000
2.2
34.3
Wash 1
480
0.0
0.4
Wash 2
3120
0.2
2.4
Elution
38880
1.9
29.6
Flow Through
Ni+-Fe #2
Ni-Fe
Purification:
96 FTU mix
IVT
15738
Flow Through
Ni+-Fe #3
IVT
Flow Through
15738
CPM Total
ug prot
% yield
BSA Block #1
1 IVT
6.5
99.9
55880
2.8
42.6
2
Flow Through
3
Wash 1
4920
0.2
3.7
4
Wash 2
2960
0.1
2.3
35160
1.7
26.8
131200
6.5
99.9
52080
2.6
39.7
5 Elution
Ni-Fe
Purification:
96 FTU mix w/
BSA
131200
BSA Block #2
1 IVT
2
Flow Through
3
Wash 1
5200
0.3
4.0
4
Wash 2
2040
0.1
1.6
30520
1.5
23.2
131200
6.5
99.9
49520
2.5
37.7
5 Elution
BSA Block #3
1 IVT
2
Flow Through
3
Wash 1
6880
0.3
5.2
4
Wash 2
2520
0.1
1.9
31800
1.6
24.2
5 Elution
Conclusions
• We have a protocol for HTP IVT protein production
with yields of >25ug unpurified material per reaction.
• Current purification protocol yields >25% (>6ug per
rxn)
• Tag positions have little effect on the polypeptide
yield, however the double tag compromises template
purity. Therefore, we propose to use the single C-tag
cassette, with second round N-tag as needed.
• All ASU protocols and electronic management
systems are in place to initiate polypeptide library
production.
T cell screening strategy effects
initial steps in library production
•Minimal demands on
polypeptide (PP) purity?
•Lower limits on amount of
input PP?
•Upper limit on PP pool
complexity?
•Multiplexed vs. linear pooling
strategy
•Complexity of arrayed PP pools
•Number of PP pools
•Scale of IVT Rxns
•Purification
Upcoming efforts and decisions
• Decision on polypeptide purification.
– We will continue to work on optimization of
protein purification protocols and integrate
new ideas.
• Decision on polypeptide pooling and assay
strategy for T cell screen.
– Before we scale up polypeptide production,
screening parameters will be solidified.
Questions after presentation
• Rick: ASU protein purification breakthrough is very important.
• Rick: UNM is working on Elispot assay for detecting response to
peptides. FT peptides may have low immunogenecity
• Stephen/Kathy: The first 500 in the pilot set of polypeptides were
selected as they likely to be very immunogenic
• Karl: use Tul 4 and spike it in to the pool?
• Kathy: Tul 4 is in the first set of 500 polypeptides as well as P11,
P12, and any in literature that were noted to be immunogenic
• Stephen: 4500 polypeptides represent the expressed FT genome.
How hard to do all 4500 on the synthesis side and use all 4500 on
the T cell assay?
• Kathy: May be better to keep separate 4500 polypeptides separate
rather than develop a pooling scheme.
• Terry: UNM can process duplicate Elispot assays using splenocytes
from naïve and vaccinated animals and can process 200 wells or 2
plates/week /person performing the assay.
Questions after presentation
• Julie: one cell concentration and only duplicate, seems a minimum
for testing the peptides
• Kathy: have to pool on a robot (not manual pipetting), could do 10
or less, then send pool 200 per week.
• Stephen: how get past the 200/week purified bottleneck
• Terry: use high throughput filter of one concentration, then titrate
other concentrations to get down. Always use naïve and vaccinated,
and an irrelevant antigen.
• Karl: host is so important, so are you using mouse? Yes.
• Rick: wants to move into nhp/human as quickly as possible.
Possibly initial screen in rat and then move into nhp and human.
Depends on the amount of protein needed in each of the 3 host
systems
• Stephen: can do elispot on rats?
• Terry: mapteck makes a rat elispot and have used at UNM
Questions after presentation
•
•
•
•
•
•
•
•
•
Rick : determining the minimal amount of protein to be used in the T cell
assay is critical at this point, then our team can better approach
pooling/mixing. Rick also definitely preferred minimal pooling. If the
potential bottleneck is a labor issue, then UNM can scale up, and test the
4500 polypeptides individually purified in the lab.
Kathy: ASI is using long polypeptides because of the multi species issuesthese polypeptides could be used with cells from mouse through humans
Kathy: 4500 polypeptides covers the whole genome
Julie: LBERI can’t pool cells from multiple non-human primates because
they aren’t inbred
Rick: Mouse isn’t as good as primates or rats, though mice would be
attractive to use for other reasons
Karl: Could we use humanized mice?
Rick: not really
Stephen: Prefers that ASU use the purified polypeptides rather than the
pooled polypeptides, as the purified polypeptides decrease competition in
the cellular assays
Kathy: With the current, improved purification scheme, ASU can really do
the production of the 4500 purified polypeptides .