Re: [ccp4bb] protein cleavage

2012-11-05 Thread R. M. Garavito
Dear Rana,

I think you need to clear up some confusion about this experiment.  MBP fusions 
suffer from a number of drawbacks depending on what you are doing.  First, did 
you use the MPB domain to purify the fusion protein (with an amylose column)?  
If so, you also purified native MBP from the E. coli as well (and there is a 
good amount in the periplasm).  Therefore you should expect to see MBP before 
and after TEV cleavage, regardless of whether you have a fusion or not.  
Second, did you see the MBP fusion on SDS PAGE, particularly on Westerns with 
anti-MBP?  Depending on your answers, we can troubleshoot your situation.

The MBP fusion vectors we have made incorporate an N-terminal His tag, followed 
by MBP and TEV, so we can purify the fusion either by Ni-chelation or amylose 
column chromatography (or both).  Also we have experienced cases where, despite 
our best efforts, MBP fusion either is a truncated expression fragment (mostly 
MBP) or has a relatively inaccessible TEV site.  For example, does DHBx 
dimerize?  This could block access to the TEV site.

But first, does the MBP-DHBx fusion exist and did you purify the fusion with an 
amylose column?

Cheers,

Michael



R. Michael Garavito, Ph.D.
Professor of Biochemistry  Molecular Biology
603 Wilson Rd., Rm. 513   
Michigan State University  
East Lansing, MI 48824-1319
Office:  (517) 355-9724 Lab:  (517) 353-9125
FAX:  (517) 353-9334Email:  rmgarav...@gmail.com





On Nov 4, 2012, at 10:24 AM, rana ibd wrote:

 Dear CCP4
  I am having a problem with cleaving my fusion protein and I would be 
 grateful if you advice me regarding this situation,  I have an MBP-DHBx 
 fusion protein and I am trying to cleave it using TEV protease, I have tried 
 different ratios and different temperatures  with different incubation time 
 but still it will not cleave, all I observe on the gel is the bands of the 
 fusion protein which is 59kDa and the MBP which is 42kDa and the TEV protease 
 which is 27kDa and no sign of the DHBx which is 17kDa,I have also checked the 
 sequence if there was any problem but I could not find anything unusual the 
 sequence was fine , so if you have any suggestions regarding this situation I 
 will be thankful
 Best Regards
 Rana



[ccp4bb] protein cleavage

2012-11-04 Thread rana ibd
Dear CCP4
 I am having a problem with cleaving my fusion protein and I would be 
grateful if you advice me regarding this situation,  I have an MBP-DHBx fusion 
protein and I am trying to cleave it using TEV protease, I have tried different 
ratios and different temperatures  with different incubation time but still it 
will not cleave, all I observe on the gel is the bands of the fusion protein 
which is 59kDa and the MBP which is 42kDa and the TEV protease which is 27kDa 
and no sign of the DHBx which is 17kDa,I have also checked the sequence if 
there was any problem but I could not find anything unusual the sequence was 
fine , so if you have any suggestions regarding this situation I will be 
thankful 
Best Regards
Rana 

Re: [ccp4bb] protein cleavage

2012-11-04 Thread D Bonsor
You do not mention what buffer you are trying to do your cleavage in. You need 
a reducing agent for TEV to work (e.g. reduced gluthionine, DTT, 
mercaptoethanol). EDTA (0.5-1mM) is recommended as TEV is a cysteine protease 
and the presence of divalent metal ions will/eventually inhibit TEV. TEV 
becomes less active as salt concentration increases (50% active at 0.5M NaCl)

If your protein contains disulphide bridges and you want to keep them intact 
you can use a ratio of 3 mM
glutathione/0.3 mM oxidized glutathione which provides enough reducing power 
for TEV to work but should not disrupt disulphides. 

If none of these reasons are why your protein fails to cleave, there is a 
strong possibility that the TEV site is inaccessible. You could try 1M urea in 
this case. TEV again will be less active, but you could at least test this 
theory.


Re: [ccp4bb] protein cleavage

2012-11-04 Thread Cynthia Kinsland
Since you're seeing the MBP band, it sounds as if you're getting some cleavage. 
 If there were no cleave you would only see the fusion and TEV bands on the gel.

It may be that your protein is not stable/soluble without the MBP fusion.  
Different buffer conditions may help if your protein is being lost 
post-cleavage.

The efficiency of cleavage may also be aided by different buffer conditions.  
Since you didn't report the buffer you're using, it's impossible to judge if it 
is appropriate for maximum TEV activity.

Cynthia

Sent from my iPhone

On Nov 4, 2012, at 10:24 AM, rana ibd 
rna19792...@yahoo.commailto:rna19792...@yahoo.com wrote:

Dear CCP4
 I am having a problem with cleaving my fusion protein and I would be 
grateful if you advice me regarding this situation,  I have an MBP-DHBx fusion 
protein and I am trying to cleave it using TEV protease, I have tried different 
ratios and different temperatures  with different incubation time but still it 
will not cleave, all I observe on the gel is the bands of the fusion protein 
which is 59kDa and the MBP which is 42kDa and the TEV protease which is 27kDa 
and no sign of the DHBx which is 17kDa,I have also checked the sequence if 
there was any problem but I could not find anything unusual the sequence was 
fine , so if you have any suggestions regarding this situation I will be 
thankful
Best Regards
Rana


Re: [ccp4bb] protein cleavage

2012-11-04 Thread SD Y

Rana,
I understood that these proteases are very efficient in cleavage. One time, I 
had a construct MBP-3C-protein, I never able to cleave this particular 
construct while I could do well with other truncations.The MBP you are seeing 
might be co-purified with DHBx and unless gel band intensity suggest that its 
coming from fusion protein. I feel cleavage site may not be  accessible for the 
protease. Might be adding few more residues might help. 
Good luckSDY

Date: Sun, 4 Nov 2012 07:24:42 -0800
From: rna19792...@yahoo.com
Subject: [ccp4bb] protein cleavage
To: CCP4BB@JISCMAIL.AC.UK

Dear CCP4
 I am having a problem with cleaving my fusion protein and I would be 
grateful if you advice me regarding this situation,  I have an MBP-DHBx fusion 
protein and I am trying to cleave it using TEV protease, I have tried different 
ratios and different temperatures  with different incubation time but still it 
will not cleave, all I observe on the gel is the bands of the fusion protein 
which is 59kDa and the MBP which is 42kDa and the TEV protease which is 27kDa 
and no sign of the DHBx which is 17kDa,I have also checked the sequence if 
there was any problem but I could not find anything unusual the sequence was 
fine ,
 so if you have any suggestions regarding this situation I will be thankful 
Best Regards
Rana 
  

[ccp4bb] protein cleavage

2012-11-04 Thread rana ibd
Dear All 
   Thank you for all your replies, the buffer for the TEV protease that I have 
used contains 50mM Tris-HCl, 150mM NaCl, 1mM EDTA, and 1mM DTT at PH= 8.0. I 
have tried using this buffer without NaCl but the TEV protease precipitates 
when dialyzing over night, as for using glutathione (reduced alone, also with 
the oxidized) still had the same result
Rana

Re: [ccp4bb] protein cleavage

2012-11-04 Thread Bosch, Juergen
@Cynthia,
On Nov 4, 2012, at 10:58 AM, Cynthia Kinsland wrote:

Since you're seeing the MBP band, it sounds as if you're getting some cleavage. 
 If there were no cleave you would only see the fusion and TEV bands on the gel.

I think that is a wrong assumption.
He did not specify if he sees the MBP band also just before TEV addition - it 
might also be truncation products which we happen to see all the time. The 
ratio varies depending on the construct but it can be as bad as a 1:1 ratio. 
You can really only tell if TEV cleaves if you do a time course experiment at 
RT with a defined amount of your protein and see if the fusion construct 
decreases. An alternative for the lack of your 17kDa desired band is simply 
your fusion construct is cleaved but your cleaved product might a) not be 
soluble at that pH or b) aggregates and precipitates.
You might be able to perform the cleavage on the Amylose column keeping a 
constant flow cycling.

Jürgen


..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry  Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu






Re: [ccp4bb] protein cleavage

2012-11-04 Thread Cynthia Kinsland
I assumed, perhaps wrongly, that the fusion was homogeneous prior to cleavage 
as the existence of the MBP band post-cleavage would not have been noteworthy 
if it had also been there pre-cleavage.

A time course or at least a time zero sample is always a good idea.

Truncation products prior to proteolysis can certainly complicate 
interpretation of results.

In my experience, precipitation of the target protein upon proteolysis from an 
MBP fusion is a fairly common problem and not necessarily fixable.


Cynthia

Sent from my iPhone

On Nov 4, 2012, at 1:19 PM, Bosch, Juergen 
jubo...@jhsph.edumailto:jubo...@jhsph.edu wrote:

@Cynthia,
On Nov 4, 2012, at 10:58 AM, Cynthia Kinsland wrote:

Since you're seeing the MBP band, it sounds as if you're getting some cleavage. 
 If there were no cleave you would only see the fusion and TEV bands on the gel.

I think that is a wrong assumption.
He did not specify if he sees the MBP band also just before TEV addition - it 
might also be truncation products which we happen to see all the time. The 
ratio varies depending on the construct but it can be as bad as a 1:1 ratio. 
You can really only tell if TEV cleaves if you do a time course experiment at 
RT with a defined amount of your protein and see if the fusion construct 
decreases. An alternative for the lack of your 17kDa desired band is simply 
your fusion construct is cleaved but your cleaved product might a) not be 
soluble at that pH or b) aggregates and precipitates.
You might be able to perform the cleavage on the Amylose column keeping a 
constant flow cycling.

Jürgen


..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry  Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu






Re: [ccp4bb] protein cleavage

2012-11-04 Thread VAN RAAIJ , MARK JOHAN
We once had a more-or-less MBP-sized fragment before cleavage, but  
this turned to be a spontaneous mutation. This expression experiment  
had been started from a glycerol stock with an unknown number of  
growth cycles prior to expression.
Starting from a fresh transformation with the purified and sequenced  
plasmid solved the problem.
Since then, I insist everybody does a fresh transformation before  
every expression experiment and not generate extra growth/dilution  
cycles beyond the normal transformation, growth on plate, overnight  
culture, dilution into large-scale expression culture.



Quoting Bosch, Juergen:


@Cynthia,
On Nov 4, 2012, at 10:58 AM, Cynthia Kinsland wrote:

Since you're seeing the MBP band, it sounds as if you're getting  
some cleavage.  If there were no cleave you would only see the  
fusion and TEV bands on the gel.


I think that is a wrong assumption.
He did not specify if he sees the MBP band also just before TEV  
addition - it might also be truncation products which we happen to  
see all the time. The ratio varies depending on the construct but it  
can be as bad as a 1:1 ratio. You can really only tell if TEV  
cleaves if you do a time course experiment at RT with a defined  
amount of your protein and see if the fusion construct decreases. An  
alternative for the lack of your 17kDa desired band is simply your  
fusion construct is cleaved but your cleaved product might a) not be  
soluble at that pH or b) aggregates and precipitates.
You might be able to perform the cleavage on the Amylose column  
keeping a constant flow cycling.


Jürgen


..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry  Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu









Mark J van Raaij
Laboratorio M-4
Dpto de Estructura de Macromoléculas
Centro Nacional de Biotecnología - CSIC
c/Darwin 3, Campus Cantoblanco
28049 Madrid
tel. 91 585 4616
email: mjvanra...@cnb.csic.es


Re: [ccp4bb] protein cleavage

2012-11-04 Thread Sangeetha Vedula
Do you see the same MBP band (or corresponding to the same MW, in case it
isn't MBP) before cleavage, at the same total concentration of protein? If
not, your protein could be crashing out. Even so, if you use SDS and heat
up the sample, I am surprised that your protein just disappeared. TEV
protease has a pretty long recognition sequence. It doesn't appear as is in
your protein, does it?

Is it possible to attach the tag at the other terminus? Perhaps it is more
accessible? Of course, it still doesn't solve the mystery of the
disappearing protein if the band is indeed MBP that appears after the
cleavage reaction.

On Sun, Nov 4, 2012 at 3:07 PM, VAN RAAIJ , MARK JOHAN 
mjvanra...@cnb.csic.es wrote:

 We once had a more-or-less MBP-sized fragment before cleavage, but this
 turned to be a spontaneous mutation. This expression experiment had been
 started from a glycerol stock with an unknown number of growth cycles prior
 to expression.
 Starting from a fresh transformation with the purified and sequenced
 plasmid solved the problem.
 Since then, I insist everybody does a fresh transformation before every
 expression experiment and not generate extra growth/dilution cycles beyond
 the normal transformation, growth on plate, overnight culture, dilution
 into large-scale expression culture.



 Quoting Bosch, Juergen:

  @Cynthia,
 On Nov 4, 2012, at 10:58 AM, Cynthia Kinsland wrote:

 Since you're seeing the MBP band, it sounds as if you're getting some
 cleavage.  If there were no cleave you would only see the fusion and TEV
 bands on the gel.

 I think that is a wrong assumption.
 He did not specify if he sees the MBP band also just before TEV addition
 - it might also be truncation products which we happen to see all the time.
 The ratio varies depending on the construct but it can be as bad as a 1:1
 ratio. You can really only tell if TEV cleaves if you do a time course
 experiment at RT with a defined amount of your protein and see if the
 fusion construct decreases. An alternative for the lack of your 17kDa
 desired band is simply your fusion construct is cleaved but your cleaved
 product might a) not be soluble at that pH or b) aggregates and
 precipitates.
 You might be able to perform the cleavage on the Amylose column keeping a
 constant flow cycling.

 Jürgen


 ..
 Jürgen Bosch
 Johns Hopkins University
 Bloomberg School of Public Health
 Department of Biochemistry  Molecular Biology
 Johns Hopkins Malaria Research Institute
 615 North Wolfe Street, W8708
 Baltimore, MD 21205
 Office: +1-410-614-4742
 Lab:  +1-410-614-4894
 Fax:  +1-410-955-2926
 http://lupo.jhsph.edu








 Mark J van Raaij
 Laboratorio M-4
 Dpto de Estructura de Macromoléculas
 Centro Nacional de Biotecnología - CSIC
 c/Darwin 3, Campus Cantoblanco
 28049 Madrid
 tel. 91 585 4616
 email: mjvanra...@cnb.csic.es