UNM TVDC - ASU Tech Call Minutes: 5/22/2007
Prepared by Barbara Griffith: 5/22/2007 & 5/29/07
Sent to ASU for review: 5/29/2007
Reviewed by Kathryn Sykes, Stephen Johnston, Mitch Magee: 6/4/2007
Distributed to NIAID on: 6/4/2007
Present: Barbara Griffith, Rick Lyons, Terry Wu, Vicki Pierson, Marlene Hammer, Joe Breen
Absent: Ann Sutton, Kristin DeBord Freyja Lynn, Stephen Johnston, Alexandre Borokov
Action Items from 3/27/07 meeting:
1. Stephen: will contact the Invitrogen Sales rep and convince that bulk ivt feed system is
the best option, rather than individual columns
2. Kathy: May be able to send at least purified and unpurified polypeptides, plus Gro El, to
UNM within a month. (approx end of June)
Action items from 5/22/07 meeting:
3. Kathy: will write the SOP for the IVT protocol and feed and email Barbara an
approximate date for SOP completion
4. Barbara – contact Julie Wilder and request that ASU’s 7 peptides be tested with primate
cells in vitro (Barbara reach Julie on 5/24/07 & Julie looks forward to receiving the
peptides)
5. Kathy/Hetal will add a 2nd His, and drop out bap/tev from LEE constructs
6. Mitch will ask Phil Stafford, who performed the initial microarray data processing, what
the cut off was. Was it 2 fold?
7. UNM/ASU will discuss the next series of mouse samples that UNM will send to ASU, off
line (completed 5/22/07 after the Sub Tech call)
8. Kathy: Make large batch of Tul 4 and Gro EL
i. Unpurified direct lysates
ii. Ivt lysates, acetone precipitate and resuspended in PBS
iii. Ivt lysates acetone precipitate and Ni/His tag purified
iv. Negative control : send lysates w/o template and also send GFP as a
negative protein as it should not stimulate T cells in vitro
The meeting was recorded for the purposes of the minutes.
1. Slide 1: Progress Report – ASU 5/22/07
a. Active Milestones: 26, 28, 33, and 34
2. Slide 2: Milestone 26 Flow Diagram
a. IVT protocols and feed are working well
b. Purification is the current issue
c. Marlene: will Kathy write SOPs?
d. Action Kathy: will write the SOP for the IVT protocol and feed
e. Barbara: time frame for the SOP?
f. Kathy: recruiting for lost employees and new one starts in 2 weeks. Will get
dates from Kathy in one month
3. Slide 3: Previous Status
4. Slide 4: Template modifications
a. His tag is more efficient than the biotinylation, and His continues to be the
better choice. Addition of the biotin to the biotinylation site is not 100%
efficient but His tag addition is 100% efficient. Elution specificity is not a
problem but elution efficiency is a challenge
b. His at both N and C ends maximizes tag exposure and allows usage of lower
urea concentration
c.
To really use biotinylation, would need to spread the biotinylation sites out
across the protein. Currently could have steric hindrances.
d. If don’t need to purify the proteins, then both His tag and biotinylation are
unnecessary.
5. Slide 5: IVT Expression Template
a. Currently making the templates with His at N and C.
6. Slide 6: Previous Status
a. Product recovered is reasonably full length
7. Slide 7: Nickel purification of 5 FTU polypeptides
a. Joe: are the purification problems unique to Ft proteins?
b. Kathy: ASU is trying to use affinity purification methods normally used on
much larger amounts of protein rather than ivt lysate solutions. An absolute
amount will remain stuck on the beads, independent of the total amount
loaded. Trying to bind and elute small amounts of proteins is harder than
binding a larger mass of protein and eluting. Stephen had used recombinant
proteins with larger masses loaded on the beads, and it had worked well.
c. Joe: have you tested with small amounts of control proteins?
d. Kathy: yes, purifying antigens on same beads, the lab gets proportional
amount lost
e. Joe: can you scale back the bead or batch?
f. Kathy: tried lowering the amount of beads, but get less sticking in column
than beads. Binding to beads is a faster method for purifying a larger number
of proteins. ASU currently can’t elute enough and consistently, relative to the
amount loaded to the beads
g. Marlene: what is the pH buffer?
h. Kathy: 0.1 N HCl, which is a low acid but is the harshest elution method,
relative to imidazole and EDTA elution
8. Slide 8: Table 1: Summary of IVT purification yields
a. “Soluble PPT in urea”: lysates resuspended from acetone and into 8 M urea
to maximize the His tag exposure to the beads
b. “Resin bound” is the percent of total that is attached to the nickel
c. “EDTA/protease solution”: elution buffer
d. “pH elution”: eluted with 0.1N HCl
e. Tracking binding and recovery with S35 label on the proteins
f. Ftu 901 had highest yield and was able to elute the most
g. FTu 1602: most stayed on the beads
h. Ftu 901 and FTU p11_ made more small products and came off the beads
better than the lysates with the longer products. These two also had the
highest yields of protein in the lysates
9. Slide 9: Table 1, cont: Summary of IVT purification yields
a. Unfolded proteins tend to be less sticky
10. Slide 10: Summary table
11. Slide 11: Conclusions
12. Slide 12: Current Status
a. 6 selected proteins are known immunogenic in T cell assays and two are
unknown lipoproteins
b. Have added Gro EL to the list
13. Slide 13: Purification of FTU polypeptides
a. “T”: total lysates (odd lanes)
b. “P”: post his purification with 1 his tag (even lanes)
c. Kathy: 901 (Tul 4) and 1419 (pll): included synthesis of partial products and
had the most amount eluted. Had highest elution efficiency
d. Kathy: wants to decrease “unfolded proteins” in their preps. Unfolded
proteins are stickier, exposing hydrophilic regions. If could lower the urea
concentration, then may also decrease incorrect folding too. Kathy wants to
add a His at both ends, so at least one partially folded protein has an
exposed His tag for binding and is less sticky so it elutes better
e. Rick: focus on purified vs. unpurified in primate assays, which are working.
Perhaps test in primates and if unpurified works well in vitro, then don’t need
purification
f. Action: Barbara – contact Julie Wilder and request that ASU”s 7 peptides be
tested with primate cells in vitro (Completed 5/24/07)
14. Slide 14: 6 FTU Pilot preps
15. Slide 15: Purification work-up Plans
16. Slide 16: Work plan for upcoming month:
a. Rick: prefers two proteins and pursue in parallel with ASU working on elution
problem. Kathy agrees with this strategy
b. Kathy: has lots of 901 and 1712 mass
c. Action: Kathy: Make large batch of Tul 4 and Gro EL
i. Unpurified direct lysates
ii. Ivt lysates, acetone precipitate and resuspended in PBS
iii. Ivt lysates acetone precipitate and Ni/His tag purified
iv. Negative control : send lysates w/o template and also send GFP as a
negative protein as it should not stimulate T cells in vitro
d. Rick: will test in micromolar quantities and do in mice and primates
e. Joe: agrees with the strategy
f. Hetal is making a set of these 3 versions above, with 2 polypeptides
g. Rick: biotin has multiple challenging problems. Should you remove the biotin
and could removing the biotin site help the His binding?
h. Kathy ;bir A is only a couple AA, so removing it would not help much.
i. Rick: thinking that removing the bap and tev, to determine if it impacts the
His binding and elution.
j. Kathy: can do this
k. Joe: agrees, add 2nd His, drop out bap /tev,
l. Action: Kathy/Hetal will add a 2nd His, and drop out bap/tev from LEE
constructs
17. Slide 17: Milestone 28 Flow Diagram
18. Slide 18: Previous Status
19. Slide 19: HTP ORF production
20. Slide 20: Work plan for upcoming month
21. Slide 21: Milestone 33 Flow Diagram
a. Mitch has performed a number of hybs with purified RNA and comparing PLL
and Corning UltraGAPs; PLL consistently works better
b. TIGR: still not released the slides and MTA has left ASU and gone to TIGR
22. Slide 22: Question: Does LAPT allow for nonbiased amplification of
Francisella RNA?
a. Got good signal intensities with the hybs
b. Got good results down to 1 ng of purified SCHU S4
c. Did hyb of amplified SCHU S4 in presence of mouse lung RNA
23. Slide 23: LAPT- GDP Confirmation
a. Struggled to reproduce the results
b. Had switched SOP for the mouse lung RNA preparations. Went from
Rneasy to Tri alone w/o Rneasy. Tri alone was leaving an inhibitor in the
RNA
c. Repeated RNA isolation with Tri , then did Rneasy. Mitch got good
amplification yields again
d. Are hybing these now
e. Tri alone is not as good as Tri followed by Rneasy
f.
Rick: UNM discovered same issue and routinely cleans up on Rneasy, after
initial Tri RNA isolation
24. Slide 24: Milestone 34: flow diagram
25. Slide 25: Question: Can we amplify Francisella RNA from
in vivo samples?
a. Used LAPT 5 protocol and got good results
26. Slide 26: Spearman Analysis of the Rank Order Between Samples
a. Ms 2= mouse 2
b. Good reproducibility across the 3 mouse samples used
27. Slide 27: Heatmap of In Vivo Expression Values
a. In vitro: lawns of bacteria are grown on plates and then RNA isolated
b. In vivo: Some genes are more highly expressed in invitro than in invivo (Ms
2,3,5) and there is a subset that are suppressed
i. The suppressed subset is 50s ribosomal unit, and glycerol
dehydrogenase , which are maintenance, housekeeping type genes
ii. Kathy: Relative to the invitro, supplemented, nutrient rich
environment, the in vivo environment is more stressful to the bacteria
so housekeeping protein synthesis goes down and stress response
proteins likely to go up. Mitch’s data shows housekeeping genes
being suppressed
c. Good efficiency of amplification and hyb to arrays well
28. Slide 28: In Vivo to In Vitro Comparison
a. In vivo up regulated, generated a list of 15-20 genes. There is a 5 genes set
within a 37 gene region. Could it be an island? Mitch grouped them into
functional categories like
i. AA metabolism or production
ii. Regulatory group- repressor of an operon
iii. Nif 3 family- transcription factor and iron binding protein- Nif 3 family
0519 is an iron binding protein; bacteria may be able to increase iron
binding protein synthesis in an environment that is iron deficient
iv. Energy metabolism
v. Thioreduction- is important in Salmonella growth in vivo; if knocked
out in macrophages, the Salmonella didn’t grow well in
macrophages or mice. Rick: makes intracellular sense
b. Rick: very reproducible between 3 different mice; patterns are similar
c. Joe: what is the cutoff with fold up and fold down;
d. Mitch: at least 2 but needs to ask Phil.
e. Action: Mitch will ask Phil Stafford who performed the initial data processing,
what the cut off was.
29. Slide 29: Percent GC Content Plot
a. Shows whole genome’s GC content; average is 32.6%
b. Blue region, might be an island, but only drops 1% which may or may not be
significant as an island. Other regions drop more than 1%.
30. Slide 30: Interpretations and Other findings
a. PLL is better than Corning UltraGaps in this test and will do more
comparisons
b. Will use Tri and Rneasy; will take Terry’s mouse infected lung RNA which
was isolate Tri reagent only. Mitch will clean it up with Rneasy and compare.
c. Rick: UNM didn’t want to manipulate the RNA from infected mouse lungs
much before sending to Mitch, initially
d. Joe: thinks TIGR slides will be useful and thanks Mitch for his patience with
TIGR. TIGR is now subsumed under J Craig Ventor institute; TIGR emails
are going away and becoming JCVI
e. Maui hyb system: chamber over top of slides and has an airbladder that
pulses to mix well over the slide. Each chamber is $20 and is not reusable.
i. Initial test: didn’t find that the Maui enhanced the hyb much
ii. Likely to send Maui hyb system back to vendor
iii. Will use Telechem aluminum chamber, that gets submerged and no
mixing
iv. Arrays are printing in quintuplicate
v. Rick: are the values the average of 5 values?
vi. Mitch: yes, but may print 3 times in future to decrease printing times.
Could reproduce the arrays in two different places on the slide too.
vii. Mitch: ASU loading 65 ul under coverslip and can’t decrease the
surface area or clearance in the chamber. The gasket size is not
adjustable.
viii. Rick: what mouse samples are needed? Action: UNM/ASU will
discuss the next series of mouse samples that UNM will send to
ASU, off line
31. Slide 31: Upcoming Monthly Transcriptome Goals
Next Calls:
1.
2.
3.
4.
5.
Prime Tech call: 5/30 Wed noon-1pm MT (2-3pm ET)- canceled
LBERI tech call: 6/5/07 Tuesday 12:00-1:00pm MT (2:00-3:00pm ET).
Cerus Tech call: 6/12/2007 Tuesday 12:00-1:00pm MT (2:00-3:00pm ET).
UTSA Tech call: 6/19/2007 Tuesday 12:00-1:00pm MT (2:00-3:00pm ET).
ASU Tech call: 6/26/2007 Tuesday 12:00-1:00pm MT (2:00-3:00pm ET).
Personnel
1. Rick away: 6/7 Thurs to 6/24 Sun
2. Barbara away: 6/20Wed to 6/25 Mon and 8/15 Wed to 8/29