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 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] [OT]: Purification in ArcticExpress

2012-09-24 Thread Cynthia Kinsland
Incubation with MgCl2/ATP/KCl facilitates the release of the Cpn60.

See: R. E. Joseph; A. H. Andreotti, Protein Expr. Purif. 2008, 60, 194–197.

Best,

Cynthia


__
Cynthia Kinsland, Ph. D. 

Director, Protein Production Facility

Cornell University

B78 ST Olin Lab

Ithaca, NY 14853-2703

Tel: (607) 255-8844

E-mail: cl...@cornell.edu


On Sep 24, 2012, at 10:38 AM, Katherine Sippel wrote:

 Hi All,
 
 I've recently swapped over to expression in the ArcticExpress(DE3) cells for 
 a particularly rock-like protein. I've got soluble expression but I'm having 
 the issue of Cpn60 (the overexpressed chaperonin) piggybacking along with the 
 tagged protein. Google-fu and the CCP4bb archives indicate that this is a 
 known issue but I haven't seen a solution thus far. Does anyone out there 
 have any tricks up their sleeve? 
 
 Also in case you were wondering in the various BL21 lines, even at extremely 
 low temperatures and 10 uM IPTG, it expresses well but produces inclusion 
 bodies that are completely insoluble in 8M urea, 6M guanidine, high 
 temperature, high pH, high reducing agent, and a number of detergents so 
 swapping back to another cell line isn't in the cards. 
 
 Any help would be appreciated.
 
 Cheers,
 Katherine


Re: [ccp4bb] His Purification

2012-01-18 Thread Cynthia Kinsland

Another option is to try NEB NiCo21(DE3) cells.  
http://www.neb.com/nebecomm/products/productC2529.asp

I have no relation to NEB beyond being a customer.

They've mutated GlmS to eliminate binding to IMAC resins and have  
added chitin affinity tags to SlyD, ArnA and Can to allow simple post- 
IMAC removal of those common contaminants.


I've just started testing them and would be interested in feedback  
from others.


Best,

Cynthia


On Jan 18, 2012, at 10:21 AM, Ed Pozharski wrote:


On Wed, 2012-01-18 at 18:26 +0530, PULSARSTRIAN wrote:

The Problem is I am not able to get rid of the infamous contamination
proteins of arnA gene (72 kDa) and glmS gene (67 kDa).


This is only a problem if you plan to have imac purification as your
only step.  If the goal is crystallization, such products can  
definitely
be used in initial trials (in fact, impurities may provide a benefit  
of

nucleation), but you would have to introduce a second step eventually,
which is often the ion-exchange.  The co-purified proteins will be
definitely removed at that step, so perhaps your time is better spent
optimizing some high-res ion-exchange gradient.

Two ways I can think of if you'd rather stick with IMAC is to try  
cobalt
instead of nickel (might have different non-specific binding  
profile) or

overload the resin with your protein (the presumption here is that it
has higher affinity than impurities and you will get rid of them by
simple competition).  Or maybe the imidazole gradient could help.

Cheers,

Ed.

--
I'd jump in myself, if I weren't so good at whistling.
  Julian, King of Lemurs




Re: [ccp4bb] TEV nucleotude sequence with restriction site

2009-06-05 Thread Cynthia Kinsland
I'm not quite sure what you want, but I have a series of vectors  
encoding various N-terminal tags and fusions, all followed by a TEV  
site.  They have an MCS standard to many pET vectors.  Therefore, they  
are designed to clone your gene in using the NdeI site at the 5' end  
(which will, after proteolysis, leave you with GH at the N-terminus of  
your protein).  Other restriction enzymes in the MCS can be used, but  
more amino acids will be left at your N-terminus.


I've used WatCut (from the U. Waterloo) for the silent mutagenesis  
question: http://watcut.uwaterloo.ca/watcut/watcut/template.php


Best,

Cynthia

On Jun 5, 2009, at 5:41 PM, Jacob Keller wrote:


Dear Crystallographers,

Does anybody have a TEV-protease-site-coding nucleotide sequence  
with a commonly-used restriction site in it, preferably right at the  
end? Alternatively, does some somebody know of a program to  
determine all equivalent codon permutations for a small coding  
region, filtered for resulting restriction site possibilities? It  
seems like it would be an easy enough script to write...


(I have already done some googling around for such a program, with  
not much luck.)


Jacob

***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
Dallos Laboratory
F. Searle 1-240
2240 Campus Drive
Evanston IL 60208
lab: 847.491.2438
cel: 773.608.9185
email: j-kell...@northwestern.edu
***


Re: [ccp4bb] problem in transformation of pqe 30 clone

2009-05-04 Thread Cynthia Kinsland
Using pQE30, any E. coli is an expression host.  Because it uses the  
T5 promoter, you don't need an E. coli strain carrying the T7 RNA  
polymerase (so, you don't need a DE3 strain).


As noted by Artem, you are most likely having a leaky expression  
problem.  However, it is odd that DH5a will transform if this is the  
case since it does not carry the lacIq mutation.  XL1-Blue does, which  
is why it was suggested below.


You could try expression right in DH5a, since you have the plasmid  
there.   Your transformation difficulties seem strange.  Using this  
vector, DH5a should not transform any more stably than the other  
strains you tried.


I second the reclone it recommendation.

Best,

Cynthia


On May 4, 2009, at 10:29 AM, Engin Ozkan wrote:

Have you tried M15[pREP4], which are the cells Qiagen would like you  
to use?  You can at least use pREP4 + your expression host, and have  
repressor expression to prevent leaky expression. That can help you  
get colonies of transformants.


Engin


On 5/4/09 7:04 AM, atul kumar wrote:


xl1-blue is not an expression host,since i have cloned it  
successfully,i need to transform into expression host, i am able to  
transform it into dh5 alfa,but not in any of expression host


From: ar...@xtals.org [mailto:ar...@xtals.org]
Sent: Mon 5/4/2009 6:32 PM
To: atul kumar
Cc: ccp4bb@jiscmail.ac.uk
Subject: Re: [ccp4bb] problem in transformation of pqe 30 clone

Hi,

You are using a pQE vector which has a T5 promoter. T5 is a native- 
like
promoter, recognized by the E. coli RNA polymerase - and this means  
that
even with lac operatorsupstream there is a huge amount of leakage  
in this
system. If your protein is even moderately toxic then you have  
issues.


Your solutions are
1) to use cells with higher levels of lac repressor (XL1-blue for  
example)

2) to re-clone this ORF under some tightly controlled promoter

Artem


 i have cloned my gene successfully into qiagen vector into pqe30  
but i do

 transformation of this into BL21,pLys,Rosseta,C41, i dont get any
 colonies,comp cells are good other clones give good no of  
colonies upon

 transformation.
 can someone help???
 thanks
 atul



Re: [ccp4bb] OT: VectorNTI alternatives - 4 Mac?

2009-01-28 Thread Cynthia Kinsland
There is GENtle which has a whole slew of tools associated with it.   
There are versions for several platforms.


http://gentle.magnusmanske.de/

If you're used to Vector NTI, it is pretty similar (and open source  
for the ambitious).



_
Cynthia Kinsland, Ph. D.
Director, Protein Production and Characterization
Cornell University Core Lab Center
Ithaca, NY 14853




On Jan 28, 2009, at 10:02 AM, Mark Collins wrote:


Hi
Anybody have suggestions for Mac OsX alternatives?
Thanks in advance,
Mark


Re: [ccp4bb] Semet in non-auxotrophic strains

2008-11-03 Thread Cynthia Kinsland
You can express Se-Met protein in non-auxotrophs using metabolic  
inhibition (Doublie. Methods Enzymol. 276, 523-530, 1997 and Van Duyne  
et al. J. Mol. Biol. 229, 105-124, 1993).


The Studier auto-induction paper (Studier, Prot. Exp. Purif. 41,  
207-234, 2005) describes an auto-induction Se-Met medium that gives  
sufficient incorporation of Se-Met for phasing in non-auxotrophs.


Good luck,

Cynthia

On Nov 3, 2008, at 12:56 PM, Jacob Keller wrote:


Dear Crystallographers,

I have been using an E coli strain called Tuner (DE3) pLacI, which  
is not auxotrophic for methionine, nor is there an auxotrophic  
analogue available. It may be that my protein can be expressed in  
other strains, but this one works well right now, and it is usually  
better to stay with what works well. Has anybody tried growing semet  
protein in non-auxotrophic strains, or are there tricks to using non- 
auxotrophs for semet protein?


Thanks,

Jacob

***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
Dallos Laboratory
F. Searle 1-240
2240 Campus Drive
Evanston IL 60208
lab: 847.491.2438
cel: 773.608.9185
email: [EMAIL PROTECTED]
***



Cynthia Kinsland, Ph.D.
Cornell University
Protein Facility Director
607-255-8844





Re: [ccp4bb] Engineering disulfide bonds

2008-02-08 Thread Cynthia Kinsland
There are strains designed to provide a less-reducing (more  
oxidizing) environment (Origami or any of it is gami derivatives  
from Novagen).


They are deficient in the thioredoxin and glutathione reductases (I  
think I recall...I didn't look it back up).


We've used them with good success for some disulfide requiring  
proteins.  However, they grow slowly and can be sickly-seeming and  
annoying to work with.


You also have the option of sending (or, attempting to send) your  
protein to the periplasm, which is a more oxidizing environment.  The  
pelB leader is available in a variety of pET vectors.  IBA markets  
several flavors of plasmid with the ompA leader sequence.  There are  
versions of the MBP that still contain its leader sequence, and the  
DsbC and DsbA fusions (available on pET vectors) are designed for  
periplasmic export.


We've made our own version of a pET plasmid with the OmpA leader  
sequence and have used it to export toxic proteins (including a  
nuclease) to the periplasm.  Generally, I've found the OmpA leader to  
be better than the pelB leader at getting proteins to the periplasm.


Best of luck,

Cynthia

On Feb 8, 2008, at 11:02 AM, David J. Schuller wrote:


On Fri, 2008-02-08 at 10:44 -0500, Kendall Nettles wrote:


Also, can I expect 100% disulfide formation  from standard bacterial
expression (assuming good geometry of the cysteines)?


No, E. coli cells are a reducing environment.

-
== 
=

With the single exception of Cornell, there is not a college in the
United States where truth has ever been a welcome guest - R.G.  
Ingersoll
== 
=

  David J. Schuller
  modern man in a post-modern world
  MacCHESS, Cornell University
  [EMAIL PROTECTED]



Cynthia Kinsland, Ph.D.
Cornell University
Protein Facility Director
607-255-8844





Re: [ccp4bb] chromatofocusing

2007-09-04 Thread Cynthia Kinsland
You might look at this paper: http://www.ncbi.nlm.nih.gov/sites/ 
entrez?cmd=Retrievedb=PubMeddopt=Citationlist_uids=12433100


They used DEAE and have given (in the supporting materials) a ton of  
useful information.


Cynthia

On Sep 3, 2007, at 4:27 PM, James Fraser wrote:


I'm forwarding this to the listserve for someone in my lab who is
having trouble sending to the list.  Replies to the list or directly
to Nat Echols ([EMAIL PROTECTED]) are greatly appreciated...

Not question about crystallography, but that's the ultimate goal:

Has anyone used a custom-made buffer for use with the PBE media  
sold by
GE?  I found a bottle of Pharmacia PBE 94 and would like to use it,  
but

if I did I would use it frequently and on a relatively large scale, so
I'd rather not pay for the (expensive and proprietary) Polybuffer.

Alternately, has anyone done something similar with DEAE/CM media?   
I've

seen references to this (although I suspect the resolution won't be as
good), but not very good descriptions.

thanks,
Nat



Cynthia Kinsland, Ph.D.
Cornell University
Protein Facility Director
607-255-8844





Re: [ccp4bb] Off topic: where to order just miniprep columns?

2007-07-05 Thread Cynthia Kinsland

There are several sources.

You can often get them from the manufacturer of your miniprep kit  
(the catalog # is written on the side of the bag they come in within  
the kit).  Many times, they don't list in their catalog that they  
will sell it separately, but you can call and get them to give you a  
price and such.  This also goes for the individual bottles of reagent  
that come in the kit (useful when somebody knocks a bottle over).  I  
have found that the bulk of the price of the miniprep kit is in the  
spin columns, so you won't save much money getting spin columns from,  
say, Qiagen.


For third party manufacturers we have used the tubes from UPrep  
(http://www.uprepusers.com/) and they have worked fine.


I've also seen the tubes (from a different third party manufacturer)  
advertised on eBay, but can't speak to their quality.


Best,

Cynthia


On Jul 4, 2007, at 1:33 PM, D. Eric Dollins wrote:


Hello,
Sorry for the off-topic question.Does anyone know a way to just
buy the spin columns from the Qiagen (or similar) PCR clean up / Gel
extraction / miniprep kits, without buying the entire kit? Our lab is
deluged with buffer components but seem to be perpetually running out
of the columns.
Thanks
Eric



--
D. Eric Dollins, Ph.D.
C266 LSRC, Research Dr.
Duke University Medical Center
Durham, NC 27710
(919) 681-1668, [EMAIL PROTECTED]



Cynthia Kinsland, Ph.D.
Cornell University
Protein Facility Director
607-255-8844





Re: [ccp4bb] Protein expression in Minimal media (M9)

2007-04-19 Thread Cynthia Kinsland
Are you adding vitamins to your M9 minimal?  RosettaBlue is thiamin  
requiring and can not grow in the absence of thiamin.  The thiamin  
requirement is so low that you can often get slow growth to a low OD  
based on residual thiamin in the cells, but you will not get robust  
growth.


Also, minimal recipes vary pretty drastically from one another, your  
system may prefer one of the other recipes out there.  Personally, I  
have never really liked the standard M9 minimal recipe.


Contrary to another poster, I found that my cells tended to grow  
better in minimal medium if I pre-adapted them to minimal by growing  
my starters in the same minimal that I intended to use (with Met  
instead of SeMet).  However, I prefer to stick with high dilutions  
(1:1000-1:100) so that may be the difference.


For years I used the MM/CA recipe from Pryor and Leiting, Protein  
Expression and Purification, 1997 v10 pg 309.  This recipe works well  
and can be adapted for SeMet growth by subbing out the casamino acids  
for a mixture of amino acids.


For the past few years I've been using the autoinduction recipes from  
Studier, Protein Expression and Purification, v41 pg 207.  I have  
found that these work fantastically well (as long as you don't need  
to express at really reduced temperature...I only use them down to 20° 
C).  There are recipes for SeMet incorporation in there as well.


Best of luck,

Cynthia

On Apr 18, 2007, at 10:34 PM, [EMAIL PROTECTED] wrote:


Hello everybody,

Sorry for an offtopic question.  I am trying to express a protein  
in M9
minimal media for Selenomet incorporation.  When grown in LB this  
protein
expressed very well and got good crystals.  Diffraction was upto 2  
A. I am
having a hard time expressing the same protein in Minimal media.   
It took
nearly 24 hours for the OD600 to reach  ~ 0.4 before inducing with  
IPTG in
minimal media and eventually got no protein expression.  It looks  
like the

cells are not growing or taking very long to grow.  The cell line I am
using is RossettaBlue (DE3) and the plasmid is pET19b based (Novagen).

It expressed poorly in BL21 (DE3) when grown in LB and thus decided  
to use

RosettaBlue (DE3).   It worked very well in LB, but having a hard time
while expresing the same in minimal media using Rosetta Blue. Has  
anybody

tried expression in minimal media using Rosetta Blue cell line?  I am
planning to try overnight induction.

Any suggestions would be greatly appreciated.

Thanks,

Manish








*
Manish B. Shah,  PhD.
Postdoctoral Fellow
Hauptman-Woodward Medical Research Institute
700 Ellicott Street
Buffalo,  NY 14203.
*



Cynthia Kinsland, Ph.D.
Cornell University
Protein Facility Director
607-255-8844





Re: [ccp4bb] protease cleavage sites

2007-03-05 Thread Cynthia Kinsland

Hi,

I'll second the TEV protease suggestion.  We use it routinely because  
it is highly specific and easy to make ourselves (and, therefore,  
cheap).  We have never seen it cut non-specifically and, since it is  
cheap, we just chuck in a bunch and let it go.


The Prescission protease is also very specific and also available for  
home preparation (it is the 3C protease...prescission is a marketing  
name).  I don't have as much experience with it, but it has behaved  
for me so far and I know that a number of people use it routinely  
with great success.


Another nice thing about having the clones around to make your own is  
that you can make the protease with the same tag that you intend to  
cut off (say, His or GST or whatever your favorite is).  Then, you  
can remove cleaved tag, uncleaved fusion protein and the protease all  
in one post-cleavage step.


In our case, we almost always have a HisTag (often as part of some  
larger fusion) so our TEV is His-tagged.  We have some of the GE pGEX  
vector for Prescission protease, so our 3C clone has GST on it.


Best of luck,

Cynthia

On Mar 2, 2007, at 5:01 AM, Rene Frank wrote:


Hi,

A non-ccp4 Q. Sorry.

I would like to use a cleavable purification tag at the N-terminus/ 
extracellular end of my membrane protein for purification. Before I  
start, I wonder if someone could recommend a particular protease  
site that I can engineer between the tag and my protein?  How about  
a proprietary cleavage system such as the PreScission protease (GE  
Healthcare)? I  would be grateful to hear success and horror  
stories in this area.


Best wishes,

Rene


Dr R.A.W. Frank, PhD
Royal Commission for the Exhibition of 1851 Research Fellow

Prof Seth Grant Lab / Genes to Cognition
Wellcome Trust Sanger Institute
Hinxton
Cambridge CB10 1SA

Work Tel: 0044 (0)1223 834244 ext. 7318
Cell No.: 0044 (0)7870 208280
===






Cynthia Kinsland, Ph.D.
Cornell University
Protein Facility Director
607-255-8844





Re: [ccp4bb] human cDNA clones?

2007-02-20 Thread Cynthia Kinsland
You can also buy cDNA from various cell lines and tissues. A few  
hundred dollars will get you enough for 50 or so PCR reactions.  If  
you have some idea where the genes are transcribed (kidney, liver,  
whatnot) you can buy tissue specific cDNA.  You can also buy pooled  
cDNA.


We've successfully amplified quite a few genes from cDNA.  It is much  
cheaper than buying individual clones and you can keep it around for  
the next, currently undecided, project.  Clones are great, but only  
good for one project.


We've used this approach for human, rat, mouse, chicken, and maybe a  
few other projects.


Google searches work well for finding vendors, though I also like to  
use Biocompare.


Good luck,

Cynthia

On Feb 20, 2007, at 4:29 AM, Miguel Ortiz Lombardia wrote:


-BEGIN PGP SIGNED MESSAGE-
Hash: SHA1

Hi Andrew,

Can you get cDNA from human cell lines? Perhaps one of your colleagues
can provide you with a few microliters. You can amplify from there all
the genes you need (provided they are transcribed in that cell line;
therefore, better use cDNA from two-three cell lines) We have followed
this approach quite successfully.

Cheers,


Miguel

Andrew Wong escribió:

Sorry a little offtopic...

We'r trying to clones a number of putative human proteins for
crystallization. Besides IMAGE clones from OpenBiosystem, is there  
any
the cheaper way of obtaining human cDNA clones? OpenBiosystem is  
okish,

~$100Cdn for each clone, but does get abit expensive when you start
getting alot.



- --
Miguel Ortiz Lombardía
Centro de Investigaciones Oncológicas
C/ Melchor Fernández Almagro, 3
28029 Madrid, Spain
Tel. +34 912 246 900
Fax. +34 912 246 976
email: [EMAIL PROTECTED]
www: http://www.ysbl.york.ac.uk/~mol/
~~ 
~

Le travail est ce que l'homme a trouvé de mieux
pour ne rien faire de sa vie.  (Raoul  
Vaneigem)

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Cynthia Kinsland, Ph.D.
Cornell University
Protein Facility Director
607-255-8844