I have expressed a human cytokine and two of its splice variants using the insect cell baculovirus system in sf21 cells. I have used a GST fusion protein for one of the mutants as it provided the best expression level and stability using this construct. I use GST beads for purification and unfortunately get co-expression with, what I found from the literature, was intrinsically expressed insect cells contaminating glutathione binding proteins. I am currently doing experiments to cleave the GST from my fusion protein which will remove both the GST tag and the contaminating protein. However, this has yet to be successful. I then used the mutant GST fusion protein in my functional assay which is a T cell proliferation assay. My problem is this:
My mutant is supposed to suppress T cell proliferation induced by the parent cytokine. I do see the suppression in a dose dependent manner but the contaminating protein also seems to be causing at least some of the suppression, as determined in a proliferation assay using the contaminating protein and parent cytokine. Has anyone had a similar problem with gst-like proteins or glutathione on T cell proliferation? In other words has anyone seen a suppression of T cell proliferation with GST?
You need to remove the GST from your protein of interest. GST can interfere with T-cell biology, how? it is unclear. Why you did not succeed removing the GST tag? It´s a simple protocol.
What other control proteins have you tried? Do other proteins, glycoproteins, aggregated proteins influence your assay and is their dose effect similar?
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