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(no subject) - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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  #1  
Old 07-16-2009, 12:29 PM
Vicky Cook
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Hello,

I am trying to produce TEV protease, and I have a few questions. First,
why is DTT added to this enzyme? I think it has to do with its being a
cysteine protease. Second, I am observing strands or strings in my
dialysis bag, when I dialyze eluate from His bind column (in 20mM
Tris-HCl pH 7.9, 500mM NaCl, 1M imidazole) into STB (10mM Tris-HCl pH
8.0, 150mM NaCl, 1mM EDTA, 0.02% sod. azide, 5mM DTT). I think it may
have to do with either the salt concentration or the DTT? BTW, is it
imidizole or imidazole? I have seen it written both ways. I think they
are two different chemicals, but I am trained as a biologist, not too
good with chemistry....

Thanks to all.
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  #2  
Old 07-16-2009, 09:01 PM
Nick Theodorakis
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On Jul 16, 8:29*am, "Vicky Cook" <[Only registered users see links. ]> wrote:

I don't know about this enzyme specifically, but in general for
enzymes that contain free sulfhydryl groups often DTT or some other
reductant is included in its storage or reaction buffer to endure that
it doesn't get oxidized.


It's not uncommon for a protein to precipitate when the salt
concentration is changed.


Imidazole. Azoles are a class of compounds with five member rings
containing nitrogen:

<http://en.wikipedia.org/wiki/Azole>

Nick


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Old 07-17-2009, 02:15 AM
DK
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In article <mailman.600.1247763243.21502.methods@net.bio.net> , "Vicky Cook" <vcook@wfubmc.edu> wrote:


Yes. Cysteine proteases require thiol group in the active site for
them to work. So DTT is added to reduce the cysteine. In reality,
it's only necessary for long-term storage. Also, substituting DTT
for 0.5-1.0 mM TCEP is highly recommended.


1 M imidazole? That's crazy. Even 8His tagged proteins elute
quantitatively at 300 mM. Some proteins just don't like very high
imidazole (In fact, I came across couple that would denature
at 200 mM and required elution with EDTA).


Chemists are crazy :-) It's imidAzole. Any 1) 5-atom ring that 2)
contains a nitrogen and 3) at least one more non-carbon atom is
called "azole". The "imid" prefix is very mysterious, though: imidazole
contains neither "amide" nor "imide" groups and there is not such
thing as "imid".

Back to the rTEV thing: we've tried almost every system for rTEV
expression. In the end, we concluded that by far the easiest one
that gives highest yeild and good purity is this:

Protein Expr Purif (2007), 55(1):53-68

Just write to Brian Fox asking for the plasmid. I am sure he will
be happy to send it to you (as he is obliged to do so, BTW).

I have modified published protocol to exclude the fansy-shmancy
ACTA-FPLC setup down to two gravity columns eluted with a
step. As far as I can tell, both the yield and the purity are the
same as in the enzyme that they originaly gave us as a gift.
We also flash-freeze rTEV (in 50% glycerol) in liquid nitrogen
for long-term storage. Upon thawing, the aliquotes can be stored
at -20C. No matter what, I am not comfortable storing an
oxidation-prone enzyme in solution at -20C for years :-)

DK


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