It sounds like list your GST construct is not binding to the column
(or very well) when the peptidase is attached. GST needs to form a dimer
to binding to the column - I suspect that your construct interferes with
dimer formation - when the peptidase is present, but when not there due
to stalled translation (rare codon?) it binds ok. The other
possibility is that when your peptidase is present it causes aggregation
- preventing binding. Maybe it depends on the concentration of your
construct - possibly dilute and slow binding might work - but am not
sure. Also - is there much of a linker between the GST and the peptidase?
Maybe others have a suggestion...
Good luck,
Ezra
On 8/29/2010 7:28 AM, Ashok Ranjan Nayak wrote:
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
