Re: [ccp4bb] Problems with an exonuclease

[Mohammad Khan]

Dear all,

I apologize that till now I was making the mistake of not sending the
replies in the common thread, but somehow only to each person individually.
I am summarizing my answers since yesterday here:

I have tried refolding it at pH 7, 7.5 and 8. I add 4mM TCEP to my urea
buffers and reduce it to 2 mM on refolding. I have tried in presence of DTT
and also in absence of any reductant.
My refolded wild type enzyme shows activity, whereas the mutant doesn't.

Yes, I have got my protein mass-speced. Didn't see any modification!

I have tried to remove the contamination by affinity and ion exchange but
with no avail. I have to try heparin-sepharose though.

I sonicate my samples in the washing steps.
I also suspected Triton X as the contaminant. But I then figured out that
TX-100 has a high absorbance at 280 nm, rather than at 260. I will surely
try the KCl and EDTA suggestions.

I have also tried purifying in absence of TX-100, but with same results!

As for running an agarose gel, I have done so but couldn't really see
anything on an EtBr gel.

I will try the suggestions of washing with other solutions as suggested.

Thank you all for the great suggestions!

On Wed, Jun 7, 2017 at 3:36 PM, Mohammad Khan 
wrote:

> Dear all,
>
> I am working with an exonuclease by refolding it from inclusion bodies
> (IBs). I tried various constructs and hosts, but couldn't get it in soluble
> form.
>
> I lyse my cells using a cell disruptor and after solubilizing IBs with
> urea, I refold the protein by rapid dilution and get an aggregate and
> monomer peak of the same on GFC. and have checked CD as well as activity,
> both of which are good.
>
> My issues is as follows:
>
> I get a high 260 nm peak while purifying it on GFC. the 260/280 ratio can
> reach upto 2. I have tried all means to get rid of watever this
> contamination is: cleaned the IBs with 1% Triton X-100, 2 M NaCl, added
> Dnase prior to lysis. I have also used methods to remove the DNA from
> protein, if that is the contaminating agent.
> I am trying to crystallize the protein with no success so far.
> Moreover, my thermofluor assays give very low fluorescence. I use Sypro
> Orange as a fluorophore.
>
> Suprisingly, a point mutation in the active site (His to Arg) gets rid of
> the issue of contamination and gives me good thermofluor curves. I purify
> the mutant also form IBs.
>
> Can someone suggest what this "contamination" may be?
>
> Thank you for your time.
>
>
>

Re: [ccp4bb] Problems with an exonuclease

[John Newitt]

Probably nucleic acids. Increase the number or volume of washes and
improve the washing of your inclusion bodies. Instead of sonication,
we use a Polytron homogenizer to resuspend the IBs pellet during
washing. This is faster and easier. Incorporate an additional
chromatography step such as Heparin-Sepharose or Cation-Exchange with
SP-Sepharose if the pI of your protein supports this. Alternatively,
try to flow your protein through DEAE or Q-Sepharose in the presence
of a moderate concentration of NaCl (e.g. 200-250 mM), where much of
the nucleic acids should be captured.

- John

On Wed, Jun 7, 2017 at 9:36 AM, Mohammad Khan  wrote:
> Dear all,
>
> I am working with an exonuclease by refolding it from inclusion bodies
> (IBs). I tried various constructs and hosts, but couldn't get it in soluble
> form.
>
> I lyse my cells using a cell disruptor and after solubilizing IBs with urea,
> I refold the protein by rapid dilution and get an aggregate and monomer peak
> of the same on GFC. and have checked CD as well as activity, both of which
> are good.
>
> My issues is as follows:
>
> I get a high 260 nm peak while purifying it on GFC. the 260/280 ratio can
> reach upto 2. I have tried all means to get rid of watever this
> contamination is: cleaned the IBs with 1% Triton X-100, 2 M NaCl, added
> Dnase prior to lysis. I have also used methods to remove the DNA from
> protein, if that is the contaminating agent.
> I am trying to crystallize the protein with no success so far.
> Moreover, my thermofluor assays give very low fluorescence. I use Sypro
> Orange as a fluorophore.
>
> Suprisingly, a point mutation in the active site (His to Arg) gets rid of
> the issue of contamination and gives me good thermofluor curves. I purify
> the mutant also form IBs.
>
> Can someone suggest what this "contamination" may be?
>
> Thank you for your time.
>
>

Re: [ccp4bb] Problems with an exonuclease

[Edward A. Berry]

On 06/07/2017 10:46 AM, Bonsor, Daniel wrote:

It will either be two things. DNA or residual Triton-X-100.


Actually Triton X-100 absorbs more at 280 than 260 - in fact the hydroxyphenyl 
in TX-100 looks remarkably like tyrosine in RNase A. I long ago gave up hope of 
resolving triton from protein and DNA by spectral curve-fitting!
eab

Re: [ccp4bb] Problems with an exonuclease

[Bonsor, Daniel]

Several further notes after contemplation, lunch and a slow day. If the protein 
is His-tagged, you could stick the unfolded protein to Ni-Resin and extensively 
wash with 8M Urea to remove DNA/Triton-X-100, elute, and then refold. This may 
be easier than multiple sonications/centrifugations. Or you could stick the 
folded protein that you have purified to the Nickel resin and wash with high 
concentrations of salt to remove DNA/Triton-X-100.

To check if is really DNA-protein complex you have prepped, you can run two 
agarose gels. Stain one with ethidium bromide and the other one coomassie 
stain. The two bands should coincide with each other (protocol for native 
agarose gel electrophoresis can be found here 
http://www.sciencedirect.com/science/article/pii/S0003269700945986).

Daniel A Bonsor PhD.
Sundberg Lab
Institute of Human Virology
University of Maryland, Baltimore
725 W Lombard Street N370
Baltimore
Maryland
MD 21201
Tel: (410) 706-7457


From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Mohammad 
Khan
Sent: Wednesday, June 07, 2017 9:37 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Problems with an exonuclease

Dear all,

I am working with an exonuclease by refolding it from inclusion bodies (IBs). I 
tried various constructs and hosts, but couldn't get it in soluble form.

I lyse my cells using a cell disruptor and after solubilizing IBs with urea, I 
refold the protein by rapid dilution and get an aggregate and monomer peak of 
the same on GFC. and have checked CD as well as activity, both of which are 
good.

My issues is as follows:

I get a high 260 nm peak while purifying it on GFC. the 260/280 ratio can reach 
upto 2. I have tried all means to get rid of watever this contamination is: 
cleaned the IBs with 1% Triton X-100, 2 M NaCl, added Dnase prior to lysis. I 
have also used methods to remove the DNA from protein, if that is the 
contaminating agent.
I am trying to crystallize the protein with no success so far.
Moreover, my thermofluor assays give very low fluorescence. I use Sypro Orange 
as a fluorophore.

Suprisingly, a point mutation in the active site (His to Arg) gets rid of the 
issue of contamination and gives me good thermofluor curves. I purify the 
mutant also form IBs.

Can someone suggest what this "contamination" may be?

Thank you for your time.

Re: [ccp4bb] Problems with an exonuclease

[S. Mohanty]

Yes, washing IB with 10 % BPER  twice with sonication makes our IB (from 
various proteins) very clean before denaturation with 6M guanidine 
hydrochloride and further processing. Smita  

On Wednesday, June 7, 2017 12:03 PM, Nicole Thomas  
wrote:
 

 I've found that washing my IB's with B-PER helps dramatically to get rid of 
any impurities.
https://www.thermofisher.com/order/catalog/product/78248

Nicole ThomasUniversity of Wisconsin, MadisonGellman Group
On Wed, Jun 7, 2017 at 11:27 AM, Jon R Sayers  
wrote:

I missed the Triton - that will be it!
On 7 June 2017 at 15:46, Bonsor, Daniel  wrote:

It will either be two things. DNA or residual Triton-X-100. When you say, 
cleaned the IBs, do you mean you sonicated the IBs, or just resuspended the 
pellet and then centrifuged again? If the latter, try sonication. I wash my IBs 
at least 4 times with the following buffers; 1. 20mM Tris, 500mM NaCl, 1% 
Triton-X-100, pH 7.52. 20mM Tris, 500mM NaCl, 1% Triton-X-100, pH 7.53. 10mM 
Tris, 1M NaCl4. 20mM Tris, 500mM NaCl, pH 7.5 By resuspension and then 
sonication. This I find removes DNA and Triton-X-100. Also, if the pellet is 
very large, you may need to increase the number of washes, volume and length of 
sonication or split the pellet up. Other things to try…1.  Change the wash 
salt to KCl and use more, (3M). I was informed that KCl is a better disrupter 
of DNA than NaCl (I stand to be corrected if this is wrong).2.  At each 
wash stage, dissolve a small amount of IBs and measure the 260/280. The ratio 
should decrease in the latter washes, if they are working.3.  Does your 
exonuclease typically contain a divalent metal? You could try adding EDTA to 
the wash steps which may help in preventing DNA stick to your protein. All the 
best! Dan  Daniel A Bonsor PhD.Sundberg LabInstitute of Human 
VirologyUniversity of Maryland, Baltimore725 W Lombard Street 
N370BaltimoreMarylandMD 21201Tel: (410) 706-7457  From: CCP4 bulletin board 
[mailto:CCP4BB@JISCMAIL.AC.UK]On Behalf Of Mohammad Khan
Sent: Wednesday, June 07, 2017 9:37 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Problems with an exonuclease Dear all, I am working with an 
exonuclease by refolding it from inclusion bodies (IBs). I tried various 
constructs and hosts, but couldn't get it in soluble form. I lyse my cells 
using a cell disruptor and after solubilizing IBs with urea, I refold the 
protein by rapid dilution and get an aggregate and monomer peak of the same on 
GFC. and have checked CD as well as activity, both of which are good. My issues 
is as follows: I get a high 260 nm peak while purifying it on GFC. the 260/280 
ratio can reach upto 2. I have tried all means to get rid of watever this 
contamination is: cleaned the IBs with 1% Triton X-100, 2 M NaCl, added Dnase 
prior to lysis. I have also used methods to remove the DNA from protein, if 
that is the contaminating agent. I am trying to crystallize the protein with no 
success so far.Moreover, my thermofluor assays give very low fluorescence. I 
use Sypro Orange as a fluorophore. Suprisingly, a point mutation in the active 
site (His to Arg) gets rid of the issue of contamination and gives me good 
thermofluor curves. I purify the mutant also form IBs.  Can someone suggest 
what this "contamination" may be? Thank you for your time.  



-- 
Best wishes
Prof. Jon R Sayers, FRSBTel: +44 (0) 114 2159552
Email:  j.r.say...@shef.ac.ukhttp://www.sheffield.ac.uk/ iicd/profiles/sayers





   

Re: [ccp4bb] Problems with an exonuclease

[Nicole Thomas]

I've found that washing my IB's with B-PER helps dramatically to get rid of
any impurities.

https://www.thermofisher.com/order/catalog/product/78248

Nicole Thomas
University of Wisconsin, Madison
Gellman Group

On Wed, Jun 7, 2017 at 11:27 AM, Jon R Sayers 
wrote:

> I missed the Triton - that will be it!
>
> On 7 June 2017 at 15:46, Bonsor, Daniel  wrote:
>
>> It will either be two things. DNA or residual Triton-X-100. When you say,
>> cleaned the IBs, do you mean you sonicated the IBs, or just resuspended the
>> pellet and then centrifuged again? If the latter, try sonication. I wash my
>> IBs at least 4 times with the following buffers;
>>
>>
>>
>> 1. 20mM Tris, 500mM NaCl, 1% Triton-X-100, pH 7.5
>>
>> 2. 20mM Tris, 500mM NaCl, 1% Triton-X-100, pH 7.5
>>
>> 3. 10mM Tris, 1M NaCl
>>
>> 4. 20mM Tris, 500mM NaCl, pH 7.5
>>
>>
>>
>> By resuspension and then sonication. This I find removes DNA and
>> Triton-X-100.
>>
>>
>>
>> Also, if the pellet is very large, you may need to increase the number of
>> washes, volume and length of sonication or split the pellet up.
>>
>>
>>
>> Other things to try…
>>
>> 1.   Change the wash salt to KCl and use more, (3M). I was informed
>> that KCl is a better disrupter of DNA than NaCl (I stand to be corrected if
>> this is wrong).
>>
>> 2.   At each wash stage, dissolve a small amount of IBs and measure
>> the 260/280. The ratio should decrease in the latter washes, if they are
>> working.
>>
>> 3.   Does your exonuclease typically contain a divalent metal? You
>> could try adding EDTA to the wash steps which may help in preventing DNA
>> stick to your protein.
>>
>>
>>
>> All the best!
>>
>>
>>
>> Dan
>>
>>
>>
>>
>>
>> Daniel A Bonsor PhD.
>>
>> Sundberg Lab
>>
>> Institute of Human Virology
>>
>> University of Maryland, Baltimore
>>
>> 725 W Lombard Street N370
>>
>> Baltimore
>>
>> Maryland
>>
>> MD 21201
>>
>> Tel: (410) 706-7457
>>
>>
>>
>>
>>
>> *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of
>> *Mohammad Khan
>> *Sent:* Wednesday, June 07, 2017 9:37 AM
>> *To:* CCP4BB@JISCMAIL.AC.UK
>> *Subject:* [ccp4bb] Problems with an exonuclease
>>
>>
>>
>> Dear all,
>>
>>
>>
>> I am working with an exonuclease by refolding it from inclusion bodies
>> (IBs). I tried various constructs and hosts, but couldn't get it in soluble
>> form.
>>
>>
>>
>> I lyse my cells using a cell disruptor and after solubilizing IBs with
>> urea, I refold the protein by rapid dilution and get an aggregate and
>> monomer peak of the same on GFC. and have checked CD as well as activity,
>> both of which are good.
>>
>>
>>
>> My issues is as follows:
>>
>>
>>
>> I get a high 260 nm peak while purifying it on GFC. the 260/280 ratio can
>> reach upto 2. I have tried all means to get rid of watever this
>> contamination is: cleaned the IBs with 1% Triton X-100, 2 M NaCl, added
>> Dnase prior to lysis. I have also used methods to remove the DNA from
>> protein, if that is the contaminating agent.
>>
>> I am trying to crystallize the protein with no success so far.
>>
>> Moreover, my thermofluor assays give very low fluorescence. I use Sypro
>> Orange as a fluorophore.
>>
>>
>>
>> Suprisingly, a point mutation in the active site (His to Arg) gets rid of
>> the issue of contamination and gives me good thermofluor curves. I purify
>> the mutant also form IBs.
>>
>>
>>
>> Can someone suggest what this "contamination" may be?
>>
>>
>>
>> Thank you for your time.
>>
>>
>>
>>
>>
>
>
>
> --
> Best wishes
> Prof. Jon R Sayers, FRSB
> Tel: +44 (0) 114 2159552 <+44%20114%20215%209552>
> Email:  j.r.say...@shef.ac.uk
> http://www.sheffield.ac.uk/iicd/profiles/sayers
>
>

Re: [ccp4bb] Problems with an exonuclease

[Jon R Sayers]

I missed the Triton - that will be it!

On 7 June 2017 at 15:46, Bonsor, Daniel  wrote:

> It will either be two things. DNA or residual Triton-X-100. When you say,
> cleaned the IBs, do you mean you sonicated the IBs, or just resuspended the
> pellet and then centrifuged again? If the latter, try sonication. I wash my
> IBs at least 4 times with the following buffers;
>
>
>
> 1. 20mM Tris, 500mM NaCl, 1% Triton-X-100, pH 7.5
>
> 2. 20mM Tris, 500mM NaCl, 1% Triton-X-100, pH 7.5
>
> 3. 10mM Tris, 1M NaCl
>
> 4. 20mM Tris, 500mM NaCl, pH 7.5
>
>
>
> By resuspension and then sonication. This I find removes DNA and
> Triton-X-100.
>
>
>
> Also, if the pellet is very large, you may need to increase the number of
> washes, volume and length of sonication or split the pellet up.
>
>
>
> Other things to try…
>
> 1.   Change the wash salt to KCl and use more, (3M). I was informed
> that KCl is a better disrupter of DNA than NaCl (I stand to be corrected if
> this is wrong).
>
> 2.   At each wash stage, dissolve a small amount of IBs and measure
> the 260/280. The ratio should decrease in the latter washes, if they are
> working.
>
> 3.   Does your exonuclease typically contain a divalent metal? You
> could try adding EDTA to the wash steps which may help in preventing DNA
> stick to your protein.
>
>
>
> All the best!
>
>
>
> Dan
>
>
>
>
>
> Daniel A Bonsor PhD.
>
> Sundberg Lab
>
> Institute of Human Virology
>
> University of Maryland, Baltimore
>
> 725 W Lombard Street N370
>
> Baltimore
>
> Maryland
>
> MD 21201
>
> Tel: (410) 706-7457
>
>
>
>
>
> *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of 
> *Mohammad
> Khan
> *Sent:* Wednesday, June 07, 2017 9:37 AM
> *To:* CCP4BB@JISCMAIL.AC.UK
> *Subject:* [ccp4bb] Problems with an exonuclease
>
>
>
> Dear all,
>
>
>
> I am working with an exonuclease by refolding it from inclusion bodies
> (IBs). I tried various constructs and hosts, but couldn't get it in soluble
> form.
>
>
>
> I lyse my cells using a cell disruptor and after solubilizing IBs with
> urea, I refold the protein by rapid dilution and get an aggregate and
> monomer peak of the same on GFC. and have checked CD as well as activity,
> both of which are good.
>
>
>
> My issues is as follows:
>
>
>
> I get a high 260 nm peak while purifying it on GFC. the 260/280 ratio can
> reach upto 2. I have tried all means to get rid of watever this
> contamination is: cleaned the IBs with 1% Triton X-100, 2 M NaCl, added
> Dnase prior to lysis. I have also used methods to remove the DNA from
> protein, if that is the contaminating agent.
>
> I am trying to crystallize the protein with no success so far.
>
> Moreover, my thermofluor assays give very low fluorescence. I use Sypro
> Orange as a fluorophore.
>
>
>
> Suprisingly, a point mutation in the active site (His to Arg) gets rid of
> the issue of contamination and gives me good thermofluor curves. I purify
> the mutant also form IBs.
>
>
>
> Can someone suggest what this "contamination" may be?
>
>
>
> Thank you for your time.
>
>
>
>
>



-- 
Best wishes
Prof. Jon R Sayers, FRSB
Tel: +44 (0) 114 2159552
Email:  j.r.say...@shef.ac.uk
http://www.sheffield.ac.uk/iicd/profiles/sayers

Re: [ccp4bb] Problems with an exonuclease

[Bonsor, Daniel]

It will either be two things. DNA or residual Triton-X-100. When you say, 
cleaned the IBs, do you mean you sonicated the IBs, or just resuspended the 
pellet and then centrifuged again? If the latter, try sonication. I wash my IBs 
at least 4 times with the following buffers;

1. 20mM Tris, 500mM NaCl, 1% Triton-X-100, pH 7.5
2. 20mM Tris, 500mM NaCl, 1% Triton-X-100, pH 7.5
3. 10mM Tris, 1M NaCl
4. 20mM Tris, 500mM NaCl, pH 7.5

By resuspension and then sonication. This I find removes DNA and Triton-X-100.

Also, if the pellet is very large, you may need to increase the number of 
washes, volume and length of sonication or split the pellet up.

Other things to try…

1.   Change the wash salt to KCl and use more, (3M). I was informed that 
KCl is a better disrupter of DNA than NaCl (I stand to be corrected if this is 
wrong).

2.   At each wash stage, dissolve a small amount of IBs and measure the 
260/280. The ratio should decrease in the latter washes, if they are working.

3.   Does your exonuclease typically contain a divalent metal? You could 
try adding EDTA to the wash steps which may help in preventing DNA stick to 
your protein.

All the best!

Dan


Daniel A Bonsor PhD.
Sundberg Lab
Institute of Human Virology
University of Maryland, Baltimore
725 W Lombard Street N370
Baltimore
Maryland
MD 21201
Tel: (410) 706-7457


From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Mohammad 
Khan
Sent: Wednesday, June 07, 2017 9:37 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Problems with an exonuclease

Dear all,

I am working with an exonuclease by refolding it from inclusion bodies (IBs). I 
tried various constructs and hosts, but couldn't get it in soluble form.

I lyse my cells using a cell disruptor and after solubilizing IBs with urea, I 
refold the protein by rapid dilution and get an aggregate and monomer peak of 
the same on GFC. and have checked CD as well as activity, both of which are 
good.

My issues is as follows:

I get a high 260 nm peak while purifying it on GFC. the 260/280 ratio can reach 
upto 2. I have tried all means to get rid of watever this contamination is: 
cleaned the IBs with 1% Triton X-100, 2 M NaCl, added Dnase prior to lysis. I 
have also used methods to remove the DNA from protein, if that is the 
contaminating agent.
I am trying to crystallize the protein with no success so far.
Moreover, my thermofluor assays give very low fluorescence. I use Sypro Orange 
as a fluorophore.

Suprisingly, a point mutation in the active site (His to Arg) gets rid of the 
issue of contamination and gives me good thermofluor curves. I purify the 
mutant also form IBs.

Can someone suggest what this "contamination" may be?

Thank you for your time.

Re: [ccp4bb] Problems with an exonuclease

[Jon R Sayers]

Dear Mohammad,
If your protein is purified from insoluble material there could be some DNA
in there though if it were stoichiometric your 26 would be >> than your
280, as the former has a much higher extinction co-efficient. A ratio  of 2
is could be RNA contamination. I'd also check the mass spec of your protein
to see if it has any unusual modification - urea is notorious for cause
carbamylation of N-terminal amino groups,  and of lysine and arginine
residues.  What that does to UV I'm not sire sure but irrespective of the
UV anomaly, I'd always get the beast mass-specced!

On 7 June 2017 at 14:36, Mohammad Khan  wrote:

> Dear all,
>
> I am working with an exonuclease by refolding it from inclusion bodies
> (IBs). I tried various constructs and hosts, but couldn't get it in soluble
> form.
>
> I lyse my cells using a cell disruptor and after solubilizing IBs with
> urea, I refold the protein by rapid dilution and get an aggregate and
> monomer peak of the same on GFC. and have checked CD as well as activity,
> both of which are good.
>
> My issues is as follows:
>
> I get a high 260 nm peak while purifying it on GFC. the 260/280 ratio can
> reach upto 2. I have tried all means to get rid of watever this
> contamination is: cleaned the IBs with 1% Triton X-100, 2 M NaCl, added
> Dnase prior to lysis. I have also used methods to remove the DNA from
> protein, if that is the contaminating agent.
> I am trying to crystallize the protein with no success so far.
> Moreover, my thermofluor assays give very low fluorescence. I use Sypro
> Orange as a fluorophore.
>
> Suprisingly, a point mutation in the active site (His to Arg) gets rid of
> the issue of contamination and gives me good thermofluor curves. I purify
> the mutant also form IBs.
>
> Can someone suggest what this "contamination" may be?
>
> Thank you for your time.
>
>
>


-- 
Best wishes
Prof. Jon R Sayers, FRSB
Tel: +44 (0) 114 2159552
Email:  j.r.say...@shef.ac.uk
http://www.sheffield.ac.uk/iicd/profiles/sayers