Slide 1
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

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
Slide 2
Completed.
High yield IVT protocols are optimized
Alternative
Purification strategies are being investigated

ML26-1 – completed
ML26-2 – completed
ML26-3 – pending a. Evaluate robustness of existing HTP Ni-based protein purification steps. b. Evaluate effect of chaperone addition on protein folding.
c. Evaluate feasibility of a thioredoxin fusion as an alternative IVT template.
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Slide 4
Current robustness of HTP IVT production and purification

Total IVT reactions - 72 (100%)
Successful IVT reactions - 49 (68%)
Successfully Ni-purified - 10 (14%)
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Slide 6
• 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 feasibility of two methods toward improving protein folding.

Slide 7
Effects of GroE and DnaK addition

Calm FTU1695 FTU0721a as is w GroE w DnaK as is w GroE w DnaK as is w GroE w DnaK
Evens – Invitrogen IVT system
Odds – Roche IVT system
Conclusion: Chaperone supplements do not have detrimental effect on efficiency of IVT reaction (prepurification)
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FTU1696a FTU0721a FTU0901 as is GroE DnaK as is GroE DnaK as is GroE DnaK
1 2 3 4 1 2 3 4 1 2 3 4
1 – total ivt
2 – flow through
3 – wash
4 - eluate
1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4
Conclusion: Chaperone DnaK improved Ni based purification yield of FTU0721a but did not increase yields from FTU 1696 or
0901.
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Effects of thio fusion on efficiency of Ni based IVT protein purification

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ORF
T7-Pro
Thio.
6-His
T7-Pro Thio.
6-His
T7-Term
T7-Term ORF

Initial Linking was inefficient
Modified PCR protocol
Modified ratios of template components
1 2 3 1 2 3 1 2 3 1 2 3
1 – FTU0922a
2 – FTU1099a
3 – FTU1204a
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Same thio fusion design in plasmid constructs for test antigens
CPV172 GFP
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IVT flow wash elut IVT flow wash elut
5x more of total sample is loaded in “elut” lane relative to “IVT” lane

• Effect of thioredoxin fusion is still under investigation. Two of two fusion templates generated high purity polypeptides, both with 20%yields. If this is robust, the approach is a significant improvement over the high variability of the non-thio carrying templates.
• Assembling of the designed thio-fusion LEEs appeared to be a challenge. This has been significantly optimized. To further optimize, a set of new overlapping primers has been ordered and will be tested this week.
• IVT expression of two identically designed plasmid cloned cassettes showed activity.
Slide 14

• A test run of a plate of FTU genes through entire HTP process from LEE production to protein purification showed that if we proceed with current protocol
~70% of genes will be successfully expressed in vitro; ~14% of samples can be Ni-bead purified.
• Supplementation of IVT reaction with chaperones doesn’t have detrimental effect on IVT production, but does not universally help polypeptides fold.
• Thio-fusions may generally improve purification step. This needs to be further tested.
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• Repeat HTP test run without acetone precipitation step and with addition of urea.
• Repeat HTP test run with thio constructs
• Run IVT templates in rabbit retic lysates
• Test polypeptides in T cell assay for cross-reacting and antigen specific activity.
Slide 16

Lysate source Yield/rxn Cost/ug
(unpurified)
E. coli ~25-30ug 50 ¢-60¢/ug
Rabbit retic ~100-300ng $33-$100/ug
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Cost vs. yield comparisons
Lysate source Yield/rxn Cost/ug
E. Coli unpur
Current purif
Possible thio pur
Rabbit retic unpurified
~25-30ug
0.25-2.5ug
~5-6ug
50 ¢-60¢/ug
$5.5-$55/ug
$2.5-$3/ug
~100-300ng $33-$100/ug
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• We will prepare all antigen samples as described above: IVT E. coli purified (+/- thio fusion) and Rabbit Retic unpurified to UNM.
• Based on antigen specific and non-specific reactivities in UNM ELIspot assays we will select a method for antigen production.
– Previous expt has shown no/low cross-reactivity with rabbit retic generated samples
– E. coli samples are less expensive to generate.
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Gray: (sub)milestone title
Red : inactive
Green : in progress
Build SCHU S4 proteome
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
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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
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Initial samples
Dose-Response of Infection
To Be Determined

•Two biological replicates of a dose response challenge
•Handled independently
•Positive correlation between dose responses
•Positive correlation between experiments
•Repeat amplifications were performed for reproducibility studies
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Comparisons of Genes Amplified between
Dose Response Experiments
Exp. 1 Exp. 2 Exp. 1 Exp. 2
10 3 131
178
121 10 5 132
173
125
10 4 100
192
109
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10 6 135
168
132

10 3
100
222
80
10 4
10 5
55
236
67
10 6
10 4 76
226
77
10 5
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10 3 10 4 10 5 10 6 NML 10 3 10 4 10 5 10 6 NML

Slide 27
•
FTT0619
•
FTT0625
•
FTT0492c
• FTT0211c
• FTT0553
•
FTT0925
•
FTT0560c
•
FTT0113
• FTT0494c
• FTT0580
•
FTT0036
•
FTT0184
•
FTT0502c
• FTT1290
• FTT1310c
• FTT0149c
•
FTT1520c
•
FTT0414
• FTT0906c
• FTT0932
0.799
0.774
0.765
0.765
0.763
0.748
0.721
0.699
0.674
0.972
0.97
0.936
0.914
0.902
0.899
0.89
0.874
0.867
0.856
0.843
o-methyltransferase family protein
ATP-dependent Clp protease subunit X transcriptional regulator, LysR family outer membrane lipoprotein conserved hypothetical protein
Methionyl-tRNA formyltransferase phosphoserine aminotransferase
Phosphopentomutase
CutC family protein conserved hypothetical protein
NADH dehydrogenase I, F subunit
Zinc-binding domain protein hypothetical membrane protein
Methionyl-tRNA synthetase
ATP-dependent metalloprotease
S-adenosylmethionine synthetase
Glutamate/gamma-aminobutyrate anti-porter
Phosphoglucomutase
DNA topoisomerase I
ROK family protein

•Data support the strategy of repeat amplification of samples for data averaging
•Normal lung material yields amplification products that bind to the arrays
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• Q-PCR validation of the hits
• Time Course Experiment
•Challenge with 10 4 SCHU S4 organisms
•Harvest 1,3,5,7 and 24 hours
•Parallel cultures in Chamberlain’s medium
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•
Kathy/Alex: will make IVT products in E coli and rabbit reticulocyte IVT systems, including a radioactive tag to confirm amount of IVT product made. These products will include the thio fusion and Ni purification. ASU will send UNM these IVT products in a couple of weeks.
•
Terry: will test these products within 2 weeks of receiving them from ASU, to determine whether cross reactivity has been reduced. Terry also will retest the rabbit reticulocyte background again.
•
Terry: is repeating chamberlains growth of SCHU S4 in liquid culture at a higher dose to get more RNA from the SCHU S4 liquid cultures. Will ship RNAs to ASU.
• Mitch: will determine where Gro EL, IglC and Kat G fall within Mitchs’ analyzed gene sets
• Mitch: will analyze down regulated genes as well as the up regulated genes, especially at early time points.
• Mitch: will perform Q PCR within the next couple of weeks to validate some upregulated genes
• Mitch: will perform microarrays on the mouse lung RNAs from the early time course of single SCHU S4 dose, next week. UNM sent RNAs to ASU previously.
• ASU/UNM: need to design next experiment of immunized vs non immunized mice
•
Does SCHU S4 look the same in spleen and liver, showing the same or different genes expressed? Could also do experiments in rats.
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