6/2/2017

2X-E0 Experiment Report - Final figure will be uploaded on Dropbox soon.

2XE0 Expt. report.pptx



5/22/2017

Bradford assay results for in-house XG-Sirt3 batches:

2nd Bradford Assay_5-22-17.xlsx



RC (4/27/2017):


From: Raj Chakrabarti



Sent: Thursday, April 27, 2017 10:33 AM



To: Sudipto Munshi



Subject: RE: New MST plan

Yes, but a few things to bear in mind:



--c-NAD: confirm that we may need to do all c-NAD expts within 2 weeks and thereafter since all powder will be consumed if anything goes wrong with long term storage no further expts will be possible.

SM: Yes, I will ask them to do all the c-NAD experiments within 2 weeks. Since I have sent them the c-NAD powder in small aliquots, they would only use 1 aliquot at a time, while storing the rest in -20 C, where it’s stable.



-in case the c-NAD Kds in expts 1a,b are way off of what you expect, insert an expt after 1 that to check c-NAD Kd to old SIRT3 without anything else, and compare it to NAD Kd to old SIRT3. confirm that there will be enough material for this.

SM: Yes, there will be enough material for this



--confirm in advance with 2bind that unless issues are encountered with c-NAD (requiring new expts like the one I mentioned above), all expts listed will be done within 2 weeks and see to it that the schedule is followed as closely as possible.

SM: Ok, I will confirm with them and follow up



Post on wiki.





From: Sudipto Munshi



Sent: Thursday, April 27, 2017 9:33 AM



To: Raj Chakrabarti



Subject: New MST plan

Dr. Chakrabarti,



We are asking 2bind to do the following experiments, in the following order:






2) Sirt3 (urea) + 23.5 mM NAM (max) + sat. Honokiol + NAD (titrated)



3) T-Sirt3 + 23.5 mM NAM (max) + sat. Honokiol + NAD (titrated)



4) T-Sirt3 + 23.5 mM NAM (max) + NAD (titrated)






1) As planned (list on wiki)



Do you approve the plan?




4/26/2017

Updated results of in-house Sirt3 concentrations, using two different Bradford assay procedures:

Updated_Sirt3 concentrations_Bradford Assay__4-26-17.pptx



4/20/2017

Plan for MST experiments (c-NAD and non c-NAD):


- ~3 mg c-NAD has been sent to 2bind, aliquoted into 3 tubes, containing ~ 1 mg each.
- The exact weights of the c-NAD in each tube has been supplied to 2bind.
- One aliquot will be used at a time, while the rest (powder) will be stored in -20 C till required.
- Based on the weight of c-NAD in the tube, 2bind will add the required amount of NB (at RT), to make 3 mM solution.
- The solution will be vortexed to mix properly and incubated in RT for 10 min to allow proper solubilization.
- It will then be c/f for 10 min at 14k x g at RT.
- The total amount of 3 mM solution will be aliquoted into 100 uL aliquots and stored at -80 C till required.


1) unlabeled T-Sirt3 + NAD (to check the influence of the label)
2) Sirt3 (urea) + 23.5 mM NAM (max) + sat. Honokiol + NAD (titrated)
3) T-Sirt3 + 23.5 mM NAM (max) + sat. Honokiol + NAD (titrated)
4) T-Sirt3 + 23.5 mM NAM (max) + NAD (titrated)
5) T-SIRT3 + sat. Honokiol and NAD (titrated)


1a) Sirt3 (old) + Ac-MnSOD + carba NAD (titrated)
1b) Sirt3 (old) + Ac-MnSOD + sat. Honokiol + carba NAD (titrated)
2a) T-Sirt3 + Ac-MnSOD + carba NAD (titrated)
2b) T-Sirt3 + Ac-MnSOD + sat. Honokiol + carba NAD (titrated)
3) Sirt3 (old) + Ac-p53 + carba NAD (titrated)
4) T-Sirt3 + Ac-p53 + carba NAD (titrated)




4/19/2017

Analysis of c-NAD solubility in New Buffer by HPLC:

c-NAD solubility in New Buffer.pptx



4/14/17

DLS and SEC figures for manuscript_4-14-17:

DLS and SEC figures for manuscript_4-14-17.docx



4/12/17

Plan for c-NAD solubility studies in new buffer (based on discussion with XG):

The following is the summary of our discussion. Please feel free to modify it. You can paste it on wiki as the record.
==========================================================================
SM and XG have discussed about the plan of the ongoing c-NAD solubility test.
SM will start to make 3mM c-NAD into new buffer first.
Peak Area (supernatant_5% DMSO HDAC) + Peak Area (pellet_5% DMSO HDAC) vs. Peak Area (supernatant _ new buffer) + Peak Area (pellet _ new buffer)
=============================================================================


4/11/17

Updated (4/13/17) List of MST experiments without c-NAD:
  1. unlabeled T-Sirt3 + NAD, to check the influence of the label
  2. Sirt3 (urea) + 23.5 mM NAM (max) + sat. Honokiol + NAD (titrated)
  3. T-Sirt3 + 23.5 mM NAM (max) + sat. Honokiol + NAD (titrated)
  4. T-Sirt3 + 23.5 mM NAM (max) + NAD (titrated)
  5. T-SIRT3 + sat. Honokiol and NAD (titrated)

These experiments can be finished in the week of 4/17



4/10/17

Task list for week of 4/10:


List of new MST experiments with c-NAD:

4-10-17_2bind MST experiments_c-NAD.docx



RC(4/7): Regarding SEC/DLS/etc, in addition to method writeup, the data should be in a doc in fig style with captions in case we want to use them in supporting info (right now the data is not presentable). Don't spend much time on it, just want it available in case we want to include at any point.
If a figure is not a suitable format, the results can be summarized in brief para(s).
4/6/2017

carba NAD solubility by HPLC results:

c-NAD solubility by HPLC.pptx

LCMS results for carba NAD: supernatant and pellet_done by Dalton Pharma:

Harpreet's email discussing results:

" As promised, we performed LCMS analysis for the Carba-NAD supernatant and pellet sent by you. Please find attached the LCMS for these samples named as XL-45-47(SUP) and XL-45-47(Pellet). These results confirm that most of Carba-NAD is still in the supernatant. A small peak for Carba-NAD is observed in pellet as well. I believe that pellet might not be thoroughly washed and it is contaminated with supernatant. From this preliminary experiment and results, I cannot predict exact concentration of dissolved Carba-NAD. I hope these results are helpful for your experiments. "

PDF file with the LCMS results:

LCMS for Carba-NAD supernatent and pellet_Dalton.pdf




3/29/17

carba NAD and NAD figure:

c-NAD and NAD_Figure.tif



3/29/17

Results of T-Sirt3 batches concentration determination using Bradford assay:

Bradford Assay_T-Sirt3_3-29-17.pptx



3/27/17

Information related to labeling in MST studies:

Info about labeling_2bind.docx

Signal test in Labelfree.pdf



3/24/17

Experimental methods for truncated Sirt3 (118-399), DLS and SEC:

Experimental Methods_SM_3-24-17.docx



3/23/17

Steps for in-house HPLC based solubility test for carba NAD:

Steps for in-house HPLC based carba NAD solubility test.docx



3/23/17

SEC results on T-Sirt3:

SEC Results_PMC-AT.pdf



3/21/17

In-house carba NAD solubility results:

3-21-17_in-house carba NAD solubility results.docx



3/20/17

carba-NAD solubility options:

3-20-17_carba NAD options.docx



3/17/17

T-Sirt3 - old and new DLS results:

3-17-17_T-Sirt3_old and new_DLS results.docx


Schedule for carba NAD solubility:

3-17-17_Schedule for carba NAD solubility studies.docx



3/3/2017

Update on purification and characterization of new batch of Sirt3 (118-399):


New info_Updated_3-13-17-New batch Truncated Sirt3 purification.pptx



2/24/17

Updated MST experiments list_2-27-17:

New MST experiments_2-27-17.docx



2/20/17

Kcat for Sirt3 and various peptides:

Kcat for different peptides for Sirt3.docx


Results of 30 min HPLC and FdL endpoint assays using truncated Sirt3 (118-399):

Endpoint assays.pptx



2/17/17

Commentary on DLS results for E-Sirt3 and T-Sirt3 and lit data about truncated Sirt3 vs full length Sirt3:

Commentary_2-17-17.docx



Initial rate experiments (using HPLC assay):


MST:


DLS and SEC:



1/31/17

Tentative schedule:

For MST and DLS, protein required = 1 x 800 ml batch. For SEC, protein required = 1 x 800 ml batch. For kcat experiments, amount of protein required is currently unknown. Would need to measure the specific activity of the truncated protein first.

Assuming all the required amount of protein (except for kcat) is ready by Friday 2/10, the following experiments can be done:


New MST experiments_1-31-17.docx







1/30/17

DLS report for Enzo Sirt3 and Truncated Sirt3:

PMC_DLS_report_E-Sirt3 and T-Sirt3.pdf



1/27/17

Truncated Sirt3 - imidazole gradient purification results + answers to questions:

Truncated Sirt3_Gradient purification + answers_Update 1-27-17.pptx



1/18/17

Linear regression analysis of Kd values for various target-ligand interactions, determined by MST and other orthogonal techniques.

Linear Regression Analysis.pptx



1/17/2017

Update:


Alliance_SEC_Quote.pdf



12/30/2016:

Update:


Purity optimization_Truncated Sirt3_Update 12-30-16.pptx

PMC_DLS_report_122816_revised.pdf



12/22/2016

Update:




12/16/2016

Powerpoint with discussion comments included in the slides:

With comments_New construct_Truncated Sirt3_Update 12-16-16.pptx


12/16/2016
Update:


New construct_Truncated Sirt3_Update 12-16-16.pptx

New tasks:





12/9/2016
Update


S3(118-399)pET21b + Rosetta2DE3 + 1mM IPTG.tif

S3(118-399)pET21b + Rosetta2DE3_IPTG titration.tif

New tasks:


Old tasks:
1) “You should identify some members of the groups that did truncated purification and determine whether any of them have left those groups. We may then contact them to assist with troubleshooting the protocol, e.g. as consultants under NDA. Send me a list of names and contacts.”
2) “Spend more time managing CRO protocols for characterization and get this work done by Dec at latest.”
3) “In the absence of truncated protein MST, current MST data may only be presentable as ratios between the values in presence/absence of modulator. The above answers from Thomas are also needed.
a) Screening colonies for new truncated Sirt3 in Rosetta 2 DE3 cells
b) Designing primers for TOPO cloning
c) Estimate % in house protein required for DLS



12/5/2016

PCR primers for TOPO cloning:

PCR primers for TOPO cloning of Sirt3(118-399).docx


12/5/2016
Updated task list:

Old tasks:
1) “You should identify some members of the groups that did truncated purification and determine whether any of them have left those groups. We may then contact them to assist with troubleshooting the protocol, e.g. as consultants under NDA. Send me a list of names and contacts.”
2) “Spend more time managing CRO protocols for characterization and get this work done by Dec at latest.”
3) “In the absence of truncated protein MST, current MST data may only be presentable as ratios between the values in presence/absence of modulator. The above answers from Thomas are also needed.”

New tasks:
a) Screening colonies for new truncated Sirt3 in Rosetta 2 DE3 cells
b) Designing primers for TOPO cloning
c) Estimate % in house protein required for DLS



12/1/2016
Different approaches being used in-house for Sirt3(118-399) construct:

1) Sirt3(118-399) + pET21b (modified): This construct is identical to that used in the Jin et al, 2009 JBC paper.


2) Sirt3(118-399) + pET151/D-TOPO: This construct is identical to that used in Steegborn, 2008 JMB paper.




11/23/2016 Update:

Sirt3(118-399) TOPO cloning schedule.docx

Updated MST data table_11-23-16.xlsx



11/18/2016 Update:


Binding data for Sirtuins.docx

MST to study enzyme substrate interactions_2bind.pptx



11/11/2016 Update:


1% Glucose experiment.tif

5 uM IPTG + 1% glucose - purification.tif



11/4/2016 Update:


Truncated Sirt3_IPTG titration.tif



10/28/2016 Update:


1mMIPTG-Induction+Lysis test.tif



10/21/2016 Update:


Truncated Sirt3_Update 10-21-16.pptx



10/14/2016 Update:




10/7/2016 Update:


1st purifn-10-7-16.tif



9/30/16 Update:


Sirt3(118-399) overexpression.pptx



9/23/16 Update:


Trucated Sirt3 overexpression.tif



9/16/16 Update on truncated Sirt3 (118-399) project:



9/9/16 Update on truncated Sirt3 (118-399) project:



9/2/16 Update:


Sirt3-Bulk batches-9-2-16.tif


Sirt3-Bulk 2-Activity-9-2-16.tif



8/26/16 Update:


Items to be included in the shipment for MST experiments.docx

Some details of the samples for MST.docx




8/24/16 - Documents related to MST experiments from 2bind:

Final MST Experimental List_PMC-AT.docx

Q1-P1-PMC Advanced Technology.pdf

Signed CDA.PDF


8/24/16 - Update on Sirt3 (102-399) Bulk batch 2 purification:




8/10/16 - Updated protein purification schedule:

Updated Protein purification schedule_SM_8-10-16.docx


7/29/16 - Update on truncated Sirt3 (118-399) :



Update 7/20/2016


Update 7/8/2016


Sirt3-AE-FPLC-Run1-7-8-16.pptx



Update 7/1/2016



Update 6/24/16




6/16/2016

Overexpression and Purification protocol for Sirt3 (102-399) from Arctic Express (DE3) cells:

Expression and Purification of Sirt3 (102-399).docx


Update 6/15/2016




AU: 6-15-2016:
I am copying and pasting the email I sent you regarding in-house Sirt3 requirement for my initial rate experiments. Thank you!
===
Hi Sudipto,
As of my current schedule, I will need total 3.36 ml of Sirt3 enzyme (assuming 1U/ul).
Here are the calculations-
Total 168 reactions for initial rate with DMSO
Total 168 reactions for initial rate with Honokiol

Total number of initial rates reactions: 336 reactions
One reaction uses 10 U of the Sirt3, so total units of Sirt3 needed = 3360 U
Assuming 1U/ul in-house
Total amount is 3.36 ml
Thank you,
Alok

==

6/10/2016

Update on tasks:



6/3/2016

Schedule for week of 6/6/16



5/27/2016

Update on peptide quantification by HPLC:


p53 peptide quantification using in-house Sirt3 (AE).ppt

RC (5/30):

- Need to see schedule for Sudipto’s work over next couple of weeks
- Including plan for truncated SIRT3 purification, preparatory work regarding other SIRT purifications, FPLC, and MST

SM (5/31):

Truncated SIRT3 purification:


Other SIRT purifications:


FPLC:


MST:


RC: Non academic or nonprofit? We are publishing our work in the open literature. Do we need to be an educational institution to access this equipment for publication of basic research in the open literature?
SM: She mentioned non-academic institutions. Her email response is below:

Dear Sudipto,

Thank you for your email and interest in working with us. Unfortunately we don’t work with non-academic institutions as a policy of our Center. Besides this, our instrument is fully book and we prioritize our Rockefeller users.
I hope you can find another MST instrument in the area.

Best wishes,

Carolina


--------------------------------
Carolina Adura Ph.D
Research Support Specialist
High Throughput and Spectroscopy Resource Center
Phone 212-327-7534
The Rockefeller University
1230 York Avenue
New York 10065
www.rockefeller.edu

Should I write to her again mentioning that we publish basic research in open literature and we intend to use their instrument for that purpose only?



Manuscript figures:


HPLC:





5/19/2016

Update on various tasks:

1) Sirt3 (102-399) purification from AE:


RC: Who will be doing the activity assays/MM initial rate studies to characterize these?
HPLC or FdL for this purpose?
SM: The activity assays by FdL has already been done for these.
RC: Initial rate MM study not same as activity study
SM: Ok, should I do the initial rate MM study with unlabeled peptides on HPLC, as you mentioned below, using the in-house enzyme?
RC: I can't answer this unless I see the complete analysis I requested on Alok's page, which includes an analysis of whether initial rate studies are possible with old HPLC.
The decision on who will do which study on HPLC will depend on who is available at that time.
I believe I have given detailed feedback to all regarding which HPLC studies are the priorities.
My question here is asking how the characterization of purified enzyme will be done immediately following each such purification, not just now but going forward.


2) HPLC:


Would you like me to do any experiments using HPLC? Please advise.

RC: You may be able to assist in establishing the detection limits of unlabeled peptides (esp at 280) other
than the p53 peptides Alok has tested so far, with old HPLC if Alok is fully occupying the new HPLC at this time.
You will need to consult with Alok in detail (see my recent posting on his page) before starting this.
It may not be warranted based on my answers to the questions regarding 214 vs 280 comparison.
If not, you may still be able to assist with some experiments on HPLC aimed at establishing the protocol for endpoint
and initial rate assays for unlabeled peptides.



3) Truncated Sirt3 (118-399) project:


4) Microscale Thermophoresis experiments:


5) FPLC setup for large-scale protein purification:


RC: To what extent do you expect the rate of your purifications to be enhanced by use of FPLC compared to your current protocol?
SM: At this point, the purifications are limited by the amount of cells I can process per column: 200 ml culture per 1 ml his-trap column, from which I obtain ~400 ul protein (Sirt3, per 200 ml batch). Doing multiple purifications this way results in different batches of Sirt3 that need to be characterized individually. Using a larger column on FPLC would increase our ability to process larger amounts of cells per purification and result in a higher amount of protein obtained after one round of purification. These larger batches of protein would be homogeneous and would require only one activity assay per large batch, thus saving time.
RC: Any idea of the extent of rate increase? Twice as fast for the 6 batches you just did?
SM: In theory, using a larger volume column (~20 ml of resin), all 6 batches could be pooled together and processed at one go.



5/13/2016

Update on Micoscale Thermophoresis expt:

Also answer the following miscellaneous question related quantity of purified protein needed for other biophysical characterization experiments:
How much protein would be needed for microscale thermophoresis expt with a modulator assuming a Kd < 100uM? How long would it take you to purify such a quantity?
Can this MST expt be outsourced to cro?

SM: Based on literature, for one MST experiment:
- pM/nM concentrations of the sample is required
- Typical volumes used per run ~4 ul
- Current purification yield of Sirt3 from AE/2M urea method = ~400 ul Sirt3 (17 - 20 uM)
- Thus, assuming an upper limit of 1000 nM concentrations of sample required per run, Sirt3 purified from one batch would be sufficient for > 2000 experiments.
- This amount of protein can be purified in less than 1 week.

- I found two CRO's that offer contract services for MST:

1) NanoTemper Technologies, Inc.
245 First Street, Suite 1815
Cambridge, MA 02142, USA
Phone: +1 650 763-1658
Fax: + 1 650 350-4390
E-mail: [email protected]
http://www.nanotemper-technologies.com/technologies/

2) 2bind
Molecular interaction services
Am Biopark 13
93053 Regensburg · Germany
[email protected]
Tel: +49 (0)941 / 943 3171 Mobile: +49 (0) 160 / 96938061

RC: Can anyone else verify this? It was previously stated that we don't have enough protein for such studies.
Please get quotes from above as well.
SM: I have written to the companies and am awaiting their response.

5/10/16

In-house Sirt3 requirements:

XG:
(1) Km, Vmax-FdL2 peptide: 42 reactions
(2) Km, Vmax-NAD+: 32 reactions
(3) Honokiol activation: 48 reactions

Total is 42 + 32 + 48 = 122 reactions
Assuming 8-10 ul of in-house enzyme is needed for one reaction, total protein needed for the above reactions is 1 – 1.3 ml.

AU:
For initial rate, to determine the Km = 700 uL of enzyme (assuming 1U/ul).

SM (5/13/16): AU's updated Sirt3 requirements: ~2.0 ml of 1U/ul enzyme.

Updated total enzyme needed:

SM: Total enzyme needed = ~3.3 ml
- Currently available = 2.5 ml
- Purification of 2 more batches required

Assuming 400 ul purified enzyme per batch, I would need to purify 4 batches.
- 2 batches tomorrow (5/11/16)
- 2 batches on Thursday (5/12/16)




5/10/16

Do you mean specific activity is comparable? Please confirm.
SM: No, only the activity in U/ul is comparable. The Enzo-Sirt3 has higher specific activity.

What do you mean by diluted sample - what dilution and why?
SM: The sample Enzo provided had an activity of 11 U/ul (as claimed on the label). This was the stock enzyme. For the reactions, the protocol asks to dilute the stock enzyme (11 U/ul) to 1 U/ul and use 5 U per reaction (5 ul of 1 U/ul enzyme). The activity reported per reaction (0.98 U/ul) reflects that of the diluted enzyme. The activity of the stock = activity of diluted enzyme x dilution factor (0.98 x 11 = 10.78 U/ul).

To achieve similar U/uL (should it be desired eg for Hplc) we would need to concentrate our sample?
SM: Yes, concentrating the sample would potentially increase the U/ul. However, since this enzyme is fragile, in the past, concentrating it led to loss of activity.

We are waiting for time estimates for purification of amounts of in house enzyme needed for the expts indicated on wiki.
SM: XG and AU will give me their approximate requirements. I am currently calculating my yields per purification batch. I will update on the time estimates once they let me know how much they need.

Regarding new protocol and other sirtuins please look into solubility issues w other sirtuins and whether there are recommended constructs, for future use. Also as time permits see if there was any indication as to why those particular truncated sirt3 sequences were used.
SM: Yes, I am looking into these issues.
SM: According to the Jin 2009 JBC paper:


Please post this and any answers to wiki.

As discussed with guan if we can make the in house enzyme fast enough it is preferable to use and esp imp to use for initial rate studies.




RC: You need to start doing some work with new HPLC on staggered hours with respect to Alok, in order to establish its suitability for initial rate assays,
which we will need to run shortly.
You should start by establishing the minimum quantifiable concentrations of the relevant components of the reaction mixture, in particular deacetylated product.
This should include the p53 peptide, not just FdL peptide.
Please discuss your schedule in this regard with Sherry.

Also see the purified protein needs of XG and AU on their respective pages.


4/19/16

Activity and specific activity results:

4-19-16-Activity-Sirt3-AE-2M urea+imidazole wash.pptx

RC: You should add slides summarizing the results from the other purification methods used in our lab and comparing the results to those
with comments.
It appears your activity in U/ul is higher, but it is not clear whether your specific activity is higher than previous methods used in our lab.
Does this suggest that your protocol has led to higher concentration of enzyme, but not necessarily less enzyme denaturation?
You should comment on this and highlight the reasons for higher concentration is it was achieved.
Regarding specific activity, since your protocol used subdenaturing urea, you should comment on why the specific activity is low.\
You should also indicate whether there are any planned pending experiments and provide their schedule.
SM: Please see updated ppt (below) with summary and comparison of previous results.
Yes both the activity and specific activity are comparable or higher than previous methods used in the lab.
No the protocol does not lead to a higher concentration of enzyme. The concentration is about the same or slightly less (0.4 - 0.6 ug/ul) as compared to previously obtained results (0.4 - 0.8 ug/ul).
The activity (U/ul) is comparable or greater than Enzo and previous protocols but the specific activity (U/ug) is lower than only Enzo. The lower specific activity can be explained by the effect of 2M urea on the enzyme (possibly some denaturation). However the purity of the enzyme from the 2M urea protocol is much higher (>85%) than Enzo (~50 - 60%).
If the current protocol / enzyme preparation is good enough for future experiments, at present, regarding the AE project, the only pending experiment is a repeat of the activity assay/std curves to check for consistency of the results. We are following the protocol sent by Enzo for the activity assay and are waiting for some items required for their protocol. If the items come in tomorrow then I can finish the repeat experiments by Friday (4/22).

UPDATED-4-19-16-Activity-Sirt3-AE-2M urea+imidazole wash.pptx

RC: Comments on the ppt:

-The old AE method provides similar specific activity to new. Provide commentary on this.
Do you suspect that 2M urea resulted in some denaturation but removal of chaperonin increased activity, compensating?
Or is it more likely that 2M urea did not denature the enzyme and chaperonin also did not affect activity?
SM: The activity/specific activity measurements previously made on Sirt3 purified by the old AE method were done using a different assay protocol. The numbers obtained for the new method were using the activity assay protocol sent to us by Enzo. Thus, only a rough comparison can be made between the two.
Yes, I think the 2M urea did result in some denaturation of the protein, thereby reducing its activity. I don't think the presence or absence of the chaperonin affects the activity as such, but its presence certainly decreases the purity. Thus, the the enzyme with chaperonin present may be expected to have lower specific activity as compared to pure enzyme without the chaperonin. However, the only way to remove the chaperonin is to use 2M urea which causes a decrease in the activity and thereby specific activity also.

-Comment on higher specific activity of Enzo in this regard (given that the old and new AE methods gave similar specific activity, w/ and w/o urea).
Could exposure time play a role?
SM: Yes, I think exposure to 2M urea definitely affects the activity, but increases the purity. Since Enzo's enzyme is impure, I think they are avoiding the use of denaturing agents (such as urea) or extensive purification methods, in order to preserve the activity.
The numbers for the old and new AE methods are only roughly comparable. I would expect the activity of the enzyme obtained using the old protocol (without urea) to be higher since the protein is not being denatured. However it's purity is very low (40-50 %), with the major impurity being the chaperonin.

-Enzo specific activity indicates that we have lost substantial activity during all of our purification protocols.
SM: Yes, the use of urea (8M for XG's protocol or 2M for my protocol) definitely reduces the activity.

-Most importantly, recall Guan mentioned another purification method from literature that required purchasing of different columns.
It was concluded, at least initially, that this method provided both high purity and high specific activity.
If possible, compare the purity and specific activities from that method to the new AE method (even rough comparison would be useful).
XG: Protein purification _reported method.pptx
SM: The Jin 2009 JBC paper describes a two stage purification: First with Ni-NTA for His-tag based affinity purification and then gel filtartion using an S200 column.
They report purities of >95 - 98%, however the number for specific activity that they report is not clear. They report a specific activity of 0.0037 in the unit of percentage of conversion of acetylated peptide substrate to deacetylated product over time and enzyme concentration.
RC: Please expand on this if possible.
SM: In this paper the author's have reported the crystal structures of two variants of human Sirt3: the full length 102-399 and the truncated 118-399. Since they are doing crystallography, they have purified both variants to >95-98% using similar protocols. Since the full length (102-399) variant is less soluble than the truncated variant, the full lenght Sirt3 was coexpressed with a chaperone protein (pG-KJE8). Other than that the overexpression and purification protocols for the two variants are similar.
The author's then compared the deacetylation activity of the two variants and found very little difference in the specific activities of the two variants: 0.0037 for SIRT3-(102–399) and 0.0033 for SIRT3-(118–399) in the unit of percentage of conversion of acetylated peptide substrate to deacetylated product over time and enzyme concentration. These numbers only represent specific activities of the two variants relative to each other and do not represent the absolute values of the specific activity.
The author's also point out that their result (both variants having similar specific activities) is in disagreement with a previous report that suggests that the activity of the truncated version(114-399) is ~50 fold higher than the full length variant (102-399).
RC: What is the meaning of "unit of percentage of conversion of acetylated peptide substrate to deacetylated product over time and enzyme concentration"? Elaborate on why you cannot express your specific activity in same units.
More importantly, Guan mentioned in ppt above that based on amount of enzyme used in those experiments, the specific activity must have been high.
Report what concentration of your enzyme would be sufficient to run the same experiments reported in this paper.
SM: In this paper, the author's have used a continuous assay based on mass spectrometric analysis of the amounts of product formed in the reaction using excess amounts of peptide substrate and NAD. Determination of the mass of the substrate peptide allows for precise determination of the degree of acetylation (i.e. substrate) as compared to deacetylated peptide (product). They first measured the initial velocities of the deacetylation reaction (Vo), as a function of time, using different (fixed) concentrations of the enzyme. For each reaction, the amount of deacetylated product was accurately determined by mass spectrometry and the Vo was expressed in units of % conversion of acetylated peptide substrate to deacetylated product over time (%/min). These values of Vo were then plotted against the respective enzyme concentrations used in the reactions (in nM) to yield linear fits. The slope of these fits (in units %/min.nM) was expressed as the specific activity of the enzyme.
The unit for specific activity that we use is U/ug, where 1 U is defined as the picomoles of substrate formed per minute, thus our specific activity is defined as pmoles of substrate fomed/min.ug. Both their's and our expressions of the specific activity are thus similar, however, since they don't specify the amount of product explicitly (in picomoles) and also express only the enzyme concentration without indicating reaction volumes (no indication of amount of enzyme in ug), their expression of specific activity is not an absolute value and is only relative to the specific activities of the other proteins calculated by the same method.
Since they use nM amounts of enzyme for their reactions, it may seem like their enzyme has high activity, but without any indication of the reaction volumes they used, it is not clear if their enzyme is indeed highly active.

Also, In their assay they are directly measuring the amounts of product formed as a function of time using mass spectrometry, whereas in ours, we are quantifying the amount of product formed relative to the fluorescent signal of known concentrations of the deacetylated standard (standard curve).

I am currently re-measuring the activity of the AE Sirt3 using the Enzo protocol. Once I have the numbers, I will try to do the above calculation.


Also answer the following miscellaneous question related quantity of purified protein needed for other biophysical characterization experiments:
How much protein would be needed for microscale thermophoresis expt with a modulator assuming a Kd < 100uM? How long would it take you to purify such a quantity?
Can this MST expt be outsourced to cro?
SM: I will try to estimate this after I have confirmed numbers for the activity.


-Regarding other sirtuins: do you have any evidence from literature that SIRT3 is more difficult to purify?
Consult with Guan and Alok to get access to wiki notes on our planned expressions and purifications of SIRT1,2.
SM: I will have to look up various protocols from literature and update on this.
SM: Based on literature, it does seem that at least the full length clone of Sirt3 (102-399) may be difficult to purify. A lot of the protocols described in literature have been tried in our lab, but have not worked. Most of the protocols don't even report estimates of purity or yield so it is difficult to ascertain how successful that protocol is. However, it does seem like Sirt3 has solubility issues (as evidenced by improved cytoplasmic yields upon coexpressing with chaperonins) which might be the reason it is difficult to purify.
I did consult XG and AU and there doesn't seem to be any planned expressions and purifications of Sirt 1,2.
RC: We discussed this to some extent before starting purification of SIRT3. We looked at SIRT1 in detail.
They have the information and it is probably also on wiki. I recall reviewing it more than a yr ago.
Now that SIRT3 purification protocol has been developed, it should be possible to make minor modifications for those.
<Pending>
SM: The purification protocols for Sirt 1,2 and 3 from literature are posted on wiki. I believe we have a Sirt1 construct from Origene, but I'm not sure if its the correct construct.


-Regarding HPLC: now that there are two HPLCs, I would like you to comment on how you might arrange a schedule
for use of 1 of them such that it does not interfere with Alok's experiments. This would initially be for purpose of
verifying on either old or new HPLC whether certain deacetylated peptide products would be detectable using these HPLCs,
and quantifying products from the new AE batch by HPLC in addition to FdL.
SM: Since Alok is not here today, I will discuss with him and comment on the schedule on Monday.
SM: Since Alok has optimized his protocol on the old HPLC, he will continue with his work on the old one. With the new HPLC, I will have to start with the following:
- Test Alok's current method on the new HPLC using the new column provided by Agilent.
- Verify resolution, retention times, etc and modify/optimize the method as required.
- Since the high concentration of BSA (1 mg/ml) in the Enzo assay buffer has caused clogging of the column and pressure problems, I will have to compare the activity of the enzyme in a different buffer (HDAC buffer from Enzo) which does not contain BSA, with the activity in assay buffer. If the activities are comparable, then I will switch over to doing all reactions in the HDAC buffer. This will help prolong the life of the column and help prevent high pressure related problems with the HPLC.

However, before I start working on the HPLC, I will have to repeat another round of purification using the 2M urea/imidazole wash method and the activity assay with AE using Enzo's protocol.

Raj




4/15/16

Update on 2M urea + imidazole wash protocols:

2M urea + imidazole wash.pptx

RC: Are you currently checking activities and specific activities after imidazole washes and if so, what is the schedule for those experiments?
SM: Yes, I am currently checking the activities of the 50 mM and 75 mM imidazole washes. I am following the protocol sent by Enzo, that they use to check the activity of their enzyme and am using the Fluoroskan instrument.
Schedule:
- Activity measurement and fluorescence standard curve (4/18)
- Concentration determination using BSA standards and calculation of specific activity (4/19)




4/11/16

Update:

- You didn't mention whether the 8M urea and old AE protocol results (purity, activity, specific activity) correspond to the final purified batches after completion of all purification steps, or whether these were also pre-imidazole wash.
SM: No, the 8M urea and old AE protocol do not correspond to the final purified batches after completion of all purification steps. The purification protocols for these two methods are different from the one that I have currently used. In both the previous two methods, an imidazole concentration of 20 mM was used in the wash step (as described in their respective protocols from the papers). In the protocol that I am using currently, there is no imidazole wash step. The wash buffer contains urea at a subdenaturing concentration to remove the Cpn60.
The protein obtained from the 8M urea protocol was >90% pure because the 8M urea denatures and removes all other contaminating proteins (and also lowers the activity of Sirt3). The protein obtained from the old AE protocol (with a 20 mM imidazole wash) was ~40-50 pure but had higher activity (no urea to denature the protein). Adding an imidazole wash step (~50 mM) to the current sub-denaturing urea protocol will most likely remove the non specifically bound proteins and increase the purity of Sirt3.


- For the previous purification methods, if the quoted numbers were following all wash steps, were the approximate purity and activity also reported pre-imidazole wash? If so, what were they? How did they compare to your current results?
SM: The approximate purity and activity for the previous purification methods were reported post-imidazole wash (20 mM). The previously quoted numbers are as follows:
For the 8M urea method - purity: >90%, activity: ~0.2 U/ul ; For the old AE method - purity: 40-50%, ~0.6 U/ul. Please note that this value for the activity is of the native protein obtained using the old AE protocol where no urea is used, thereby not damaging the protein in any way. These numbers for both of the previous methods were calculated relative to Enzo Sirt3 which had a specific activity of 1.5 - 2.5 U/ug.
For my current results - purity: 40-50%, activity for 1M wash ~0.56 U/ul, activity for 2 and 3M wash ~0.14 U/ul. The purity of all three (1,2 and 3M) are similar except for the Cpn60 contaminant, which is higher (~40%) in case of 1M and lower (~10%) in case of 2 and 3M.


- From previous discussions I recall it was claimed that the old AE protocol gave significantly higher activity and specific activity than 8M urea. Should I conclude that significantly higher meant 2.5 fold? Provide all relevant data.
SM: Yes, significantly higher meant ~2.5 fold.
The specific activity from the 8M urea protocol was 0.4 U/ug and that from the old AE protocol (with no subdenaturing urea wash) was 0.98 U/ug. These numbers were calculated relative to Enzo Sirt3 which had a specific activity of 1.5 - 2.5 U/ug.






4/8/16

Updated results of Sirt3 purification from AE, including activity data:

4-8-16-Updated Results of SIRT3-AE-urea wash.pptx



3/29/16

Updated results of Sirt3 purification from AE (DE3) cells, using sub-denaturing urea wash protocol:

Updated Results of SIRT3-AE-2M urea wash 3-29-16.pptx

RC: Please provide estimate of purity in % so we can compare to previous purification protocols.
SM: At present the purity of Sirt3 is ~50% from the 2M urea wash and ~40% from the 1M urea wash. The Cpn60 contaminant is <10% after 2M urea wash and ~20% after 1M urea wash.
RC: Is this with or without imidazole wash?
I thought the previously reported purity of AE without urea was 40-50%, with the Cpn60 band contributing a large part of the impurity?
SM: This is without imidazole wash. I followed the purification protocol exactly as described in Belval et al (2015), as a first attempt to remove the Cpn60 contaminant. Now that I see that the 2M urea wash (as described in the paper) works to remove Cpn60 to a large extent, I can optimize the imidazole wash step to remove the other contaminating proteins that are non-specifically bound to the column.
Yes, the previously reported purity from AE was 40-50%, with the Cpn60 band contributing a large part of the impurity. However there were additional contaminating bands, similar to my results. Also the previously reported results were done following a different purification protocol, with an imidazole wash step.

3/21/16

Results of Sirt3 purification from Arctic Express (DE3) cells, using 2M urea wash protocol:

Results of SIRT3-AE-2M urea wash 3-21-16.pptx

RC: Ok good preliminary results. Please repeat at the larger scale you mentioned to get clearer bands. Please indicate which steps under A,B below you would need to start at.
Also, following up on my comments below and also given the preliminary results above, please adjust the schedule to do specific activity studies on the
1-3M urea wash series before any other protocol modifications.We would like to get purity and specific activity data for these washes before considering any protocol modifications.

SM: I would have to start at step B, point 3 to repeat the experiment at a larger scale.
I currently have enough cells to repeat the 2M urea wash expt. and do the 1M and 3M urea wash experiments, after which I will have to grow more cells.
I will try to finish the different purification protocols by Thursday (3/24) and hopefully measure the activity on Friday (3/25).



3/8/16

Schedule for SIRT3 purification experiments from Arctic Express (DE3) cell line.

A) Transformation and selection of colonies for overexpression:

B) Large scale cell growth and overexpression of SIRT3:

RC: Ok, so your first purification attempt is starting about 1 week after XG's purification.
After the purification protocol is established, the plan would be to carry out analogous initial rate experiments
to those on her page on Fluoroskan. I believe you have confirmed that your scale of expression and purification (ignoring purity level at this time)
you are working with would likely be sufficient for the experiments she listed there, given what we know about AE yield and activity levels.

RC (3/15): Please confirm that you are on schedule for preliminary purification attempt with new protocol and if so please update shortly with a rough
schedule for the optimization of subdenaturing urea.

SM (3/15): Yes, I am on schedule for the preliminary purification attempt with the new protocol.
Rough schedule for the optimization of subdenaturing urea protocol - With the goal of removing the Cpn60 contaminant:

Can we please schedule a meeting to discuss the above mentioned tasks?

RC: Ok, I thought you had mentioned there was no need to meet regarding the 2M urea wash experiment. I had inquired about that recently.
If you have any questions please post here and we can meet if needed.
Or, are you referring to discussion of the heat-denatured lysate method?
The detailed comments I posted regarding subdenaturing urea method on the wiki are on "PMC Group Meeting" page.
Note also that as mentioned in those notes, that 1M urea gave 95% removal of chaperonin band in the case of the enzyme studied
in the literature.

SM: Yes, I was referring to discussion of the other optimization protocols in case the 2M urea wash method doesn't work.
Ok, I will refer to your comments.

RC: Is there any way to carry out a series of [urea] in parallel to identify the optimal subdenaturing concentration?
Please let me know if there additional reagent purchases are required for heat-denatured lysate method.

SM: Yes, in the week of 3/21, for the optimization, I will do a comparative study of 1M - 3M urea in the wash buffers.
No, at this point additional reagent purchases are not specifically required for the heat-denatured lysate method. However, we are almost out of the staining solution for the protein gels.

RC: Ok as I understand preliminary results with 2M urea will be presented on Fri. Are you planning to check specific activity as well as purity by 3/18 or will some of that be done
wk of 3/21?

Assuming we do not get suitable purity and/or specific activity at 2M, please provide a more detailed schedule for 3/21 comparative study of 1M-3M urea wash including
specific activity analysis.

Is it realistic to assume all subdenaturing urea optimization (1M-3M) as well as heat-denatured lysate,
and combination of subdenaturing urea and heat-denatured lysate can be done in week of 3/21?
Depending on schedule for subdenaturing urea optimization, we will consider when further planning of the latter is required.

SM: I am only planning to check the purity of the enzyme purified by the 2M urea method, as compared to the control, by Fri 3/18. I will run gels to analyze each purification step and the purity of the elutions.
RC: Please report this in a ppt on Fri.
SM: I finished the experiments and ran the gel, but unfortunately the bands are too faint. I need to let the gel stain longer to be able to quantify the bands. I am leaving the gel to stain over the weekend and hopefully I will be able to take a good picture of it on Monday.
RC: It may be advisable to do specific activity measurements with 1-3M urea week of 3/21 and to do heat-denatured method following week, given the discussion and above and esp given the slight delay in assessment of purity from 2M urea.
I will advise after seeing the purity results but please consider the schedule for this revised plan next week should we proceed this way.
There is also a pending task regarding JBC protocol.




I am not planning to measure the specific activity of the elutions right away. I will freeze samples obtained from each method and then measure activities of all the samples together in one experiment. That way I can directly compare activities from each method without the problem of inter-day variations in the measurements.

Week of 3/21:


Realistically, only the purification experiments and analysis of the purity of the elutions using the 2M urea, [urea] optimizations and the heat-denatured lysate methods can be done in the week of 3/21.
I can try the combination of subdenaturing urea and heat-denatured lysate method in the week of 3/28.

RC: Yes, but how would you know if you need to try the other methods until you verify whether the specific activity of the urea-only approach is sufficiently high?
Also, there is the issue of planning the detailed protocols of the other methods.

SM: From the gel analysis of the 2M urea method, I would know if the eluted protein is pure or not (i.e. if the Cpn60 contaminant has been removed); if the contaminant is still present, then I would have to try a higher urea wash (3M); if the contaminant is removed by the 2M urea wash, then I would try 1M urea to see if a lower [urea] is sufficient to remove the contaminant.
Since we are operating under the goal/assumption that high purity would also lead to high specific activity, I am currently aiming to identify the protocol which results in highest purity.
RC: If urea concentration is too high, it's not clear whether that will be the case (e.g., see 8M urea).
SM: Yes, that is why I don't want to go over 3M urea. If the contaminant is not removed by a maximum of 3M urea, then the subdenaturing urea approach (at least by itself) may not be feasible.

Yes, the heat-denatured lysate protocol needs some planning.





SM: Yes, I plan to try one purification attempt on 3/17 using the new 2M urea protocol from one batch of cells that I will grow.

So, at this time, should I focus on solving the purity problem or should I focus on purifying sufficient quantity of impure SIRT3 (SIRT3 + Cpn60 contaminant) for the initial rate experiments?
Please advise.

RC: I thought he activity of AE is expected to be at least as high as urea at the higher purity level that might be achieved by new protocol. Is this correct?
If this is the case, operate under the assumption that the protocol will work on the first try (ie no wasted protein from optimization) and tell me whether you would then have enough for all planned expts.

In order to stay on schedule in case the new protocol does not work on the first try, you may need to put in some extra work to purify the additional needed batches rapidly. We will discuss further at that time.

SM: Yes, the activity of impure SIRT3 that was previously purified from AE, was higher than that of the pure SIRT3 purified by the 8M urea method.
The current assumption is that the new protocol may improve the purity of the AE enzyme without decreasing its activity.

Since I am growing 4 x 200 ml cultures, and according to XG's calculations, protein purified from 3 x 200 ml cultures should be sufficient for the experiments, assuming the protocol works on the first try at a small scale, I should have enough batches of cells to purify enough protein for all the planned experiments.



3/3/16

Update on DMSO task list

SM: Comparative fluorescence standard curves using the FDL Deacetylated Standard were measured on the Fluoroskan Ascent FL system, with and without 5% DMSO in the reactions.
- Results suggest no major difference in the signals obtained with and without DMSO. Please see the attached powerpoint slides for the plots.
- Will be repeating one set of experiments tomorrow to see the inter-day variations in the data.
- Will also be helping XG with looking up articles for concentration ranges of SIRTs used in assays.

FDL std curve with DMSO.pptx
RC: How does this compare to Tecan?
SM: I haven't yet had a chance to compare this data with Tecan data. I will make a comparison once I have repeated one set of today's experiments tomorrow.


2/26/16

SM: Update on DMSO task list

- Experiment design done.
- Still need to get more familiar with the Fluoroskan instrument.
- Was waiting for new substrate and enzyme to come in for the set of experiments: A (below), when I moved on to purifying more enzyme.


2/11/16

SM: Updated schedule

- As asked, I will be purifying more batches of SIRT3 using the urea method for XG's and AU's experiments.
- Since this task is a current priority, I'm putting this task ahead of the Initial rate determining experiments.



2/8/2016

SM: Schedule for DMSO experiments using FdL assay

A) Initial rate determination using Enzo-SIRT3 and different concentrations of NAD+:

- [NAD+] = 375, 750, 1500, 3000 uM
- [Substrate] = 100 uM
- SIRT3 = 2.5 U/rxn
- Time points = 0, 5, 10, 20, 30, 60, 120 min
- Total reactions = 4 concentrations of NAD+ x 8 time points (including blank) = 32 reactions
- Time required = 1 day to design experiment and get hands on with the Fluoroskan instrument.
2 days to do the experiments and standard curve
2 days to analyze data and improvise plan, as required.

B) Effect of DMSO on SIRT3 activity and kinetics:

- The above experiments will be repeated using 5% DMSO in the reactions
- Time required for experiments, standard curve and data analysis = 4 days

C) Effect of DHP 1c on SIRT3 activity and kinetics:

- The above experiments will be repeated using 50 uM DHP 1c (in 5% DMSO, final conc.) in the reactions.
- Time required for experiments, standard curve and data analysis = 4 days





1/29/16**

SM: Status of learning up FdL assays:


- Currently measuring the concentration of the combined batches of SIRT3 and will subsequently measure it activity.
- Next week I will be working on the report for the latest Sirtuin assays.

RC: Has the FdL characterization included any kinetic characterization?
Is there a plan for doing that in the future after purification?

SM: The FdL assays I have done so far are the end point activity assays.
I plan to do the kinetic characterization with the SIRT3 that I will purify from the Arctic Express cells.



01/22/16

SM: The activity of SIRT3 purified in Batch 2 is identical to XG's preparation, so she will use all of the Batch 2 protein for her experiments. The activity of Batch 1 SIRT3 is slightly lower so that batch will not be used.
I am currently measuring the activity of Batch 3 protein and if the activity is again identical to XG's then she will combine Batches 2 and 3 protein for her experiments.
Next week I will be purifying a new batch for AU's HPLC experiments.





01/12/16

SM: The activity of the SIRT3 that I purified is comparable to XG's, so she can use that protein for her experiments. I will be starting a fresh round of purification tomorrow.




SM: 01-06-2016:
Protein Purification protocol- Purification protocol for recombinant SIRT3 from BL21(DE3) cells.docx
====
Re-post:
Tasks accomplished upto 1/5/16
RC: Ok, please do update on specific activity and indicate whe
SM: I did the activity assays using 500 uM NAD+ and 500 uM Pep-AC, as recommended by XG. The activity assays done by XG have 1.5 mM NAD+ and 100 uM Pep-AC. So at this point the assays are not comparable. I will be doing the assay again on Monday using XG's assay method (1.5 mM NAD+ and 100 uM Pep-AC) and also use some of her purified SIRT3 for the assay so that I can make a comparison with the SIRT3 I purified. XG-do you have any comments?


RC: Please organize tasks in reverse chronological order.
Alok, please discuss with XG the amount of protein that will be needed for the upcoming initial rate experiments and comment on when it would be ready.
AU: Does XG needs more in-house protein? If so, Sudipto is still in initial phase of determining activity, once he confirms the activity then we can discuss if Sudipto needs to do more purifications. For me, I would certainly need more protein to optimize some of the parameters as my assay is completely different that XG's.
RC: Yes, you need to talk to XG about the protein needs - I believe she has indicated she will be using some of the newly purified protein from work you guys are currently doing, for the upcoming initial rate experiments.


========


Tasks accomplished up to 12/22/2015




RC: How does the yield compare to what we obtained before?
SM: My yield of SIRT3 using the urea method is similar to that obtained before.

Tasks accomplished up to 12/17/2015


RC: What is the purification protocol described in Kumar?
SM: Please see the attached word document describing the purification protocol.
RC: Ok, so you mean the urea protocol.
SM: Yes, the urea protocol.

Tasks accomplished up to 1/5/2016



RC: Please organize tasks in reverse chronological order.
Alok, please discuss with XG the amount of protein that will be needed for the upcoming initial rate experiments and comment on when it would be ready.