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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 salt concentration to 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 40mM in the elution buffer with two different pH at 8 and 9.
I also read from the literature from similar proteases the behavior is unaltered even after doing mutagenesis of the Cysteine residue at its active site .Should I try ion exchange or affinity chromatography using any inhibitor of this enzyme??