I worked with several viral cystene proteases. I do not recommend using
basic pH for them dring purification or storage or crystallization. Keep it
below 7, because the active site cysteine oxydazes very easily. The higher
the pH, the easier the oxydation. PH 10 can also cause hydrolysis of some
peptide bonds. Try His tag, then you don't need to use proteases for
cleavage, you can use Ni column.
Maia
----- Original Message -----
From: "Ashok Ranjan Nayak" <ashokgocrac...@gmail.com>
To: <CCP4BB@JISCMAIL.AC.UK>
Sent: Sunday, August 29, 2010 5:28 AM
Subject: [ccp4bb] Query regarding GST fusion protein purification
Hello one and all !!
I have been working on cloning and purification aspects on a Leishmanial
cysteine peptidase (pI= 8.2) for quite some time. I had cloned it in pGEX
expression vector. Expression seemed quite okay when induced with 0.5 and
1mM IPTG, so did the solubility in 4 buffers at different pH conditions. i.
e. Tris (6.8), HEPES (7.5), Bicine (pH = 9) and Glycine (pH=10). The
problem is when I purify it using glutathione sepharose column I get only
GST (size wise estimation; no western tried ) i.e. a prominent 25 KD band.
At the same time I get the fusion protein in the load, equilibration and
wash fractions. When I increased Nacl concentration upto 400 mM I only
could exclude the fusion protein band from wash. I had tried protease
inhibitors like PMSF, sigma cocktail, and DTT in the lysate before
sonication. I had also tried reduced glutathione upto 40 mM in the elution
buffer with two different pH at 8 and 9.
I also read from literature that similar intracellular cysteine
proteases behave same even after mutating the conserved cysteine residue at
its active site. They all say that its not because of autocatalytic property
of the enzyme its because of some proteases from E.coli.
Should I try ion exchange or affinity chromatography using any inhibitor of
this enzyme??
Can anyone suggest me some tip?? Guys help me out. i am kind of struck here
Ashok Ranjan Nayak
Research Scholar
Molecular and Structural Biology Division
Central Drug Research Institute,
Lucknow
