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