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Fusion protein problem

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#1 cellsignaling



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Posted 18 March 2010 - 05:20 PM


I tried to fused one enzyme with its regulator protein.
I fishedout enzyme from pGex vector, amplified then ligated in pET28 vector already containing regulatory protein. I used EcoRI Digestion as pET28 was having EcoR1 at the end and finally the stop codon, so I did cut here and joined my enzyme through primer having EcoRI and hexa gly linker.

So what its gona produce will be: His6-Regulatory protein-EcoRI-6Gly-Enzyme-EcpRI.

I confirmed the orientation of my subcloned vector through Sequencing.
-Problem is that when I expressed the protein I am getting too much of my regulator protein(previously present in the same vector) and very less fusion protein. as I did confirm it with western blotting.
i was really confused and checked the sequence of my subcloned pET28 again and again to confirm if I am right and yes I am right...
Why I am getting vrey less fusion protein and more of my regulatory protein which was already there as HIS6 tagged in pET 28????
My boss suggested me that you introduced EcoRI with Glycine linker which codes for Phe-gly-gly and chymotrypsins are doing their job and cleaving most of the fused protein? when I tried to find my enzyme in case of cleavage I never got it rather a very light signal was observed at the end of the gel(might be the degraded product?????) but I observed both the regulator and enzyme blots at the same position suspected to be the fusion protein place on SDS PAGE but in very less quantity as compare to the regulator protein.

my supervisor suggested me to try Quick mutation at EcorI linked with Glycine linker and repeat it.

I need suggestions, I hope I wont confusing in explaining myself?

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