Re: [ccp4bb] Problems with an exonuclease
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
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
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
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
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
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
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
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
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
[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.
