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GST fusion protein expression


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#1 biochemist_78

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Posted 19 May 2010 - 03:19 AM

Hello everybody,
I have been struggling to express various constructs which was cloned in the pET41a vector. The length of the constructs are typically 700bp(kin4), 300bp(kin4.1) & 400bp(kin4.2). I have previously used this vector for different constructs and it worked fine. I am using 100mM of stock IPTG (1ul/ml of the culture) to induce and I have tried inducing at various temp 4'C, 15'C, 25'C & 37'C and I could not get any of them to work.

I don't know why is it doing so because I have expressed this construct in the pMal vector from which I assume the proteins being expressed should not be toxic to the cell.

If anybody can please help me out, I would really appreciate it.

#2 MERCIFULJ

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Posted 31 May 2010 - 07:51 PM

Hello, have you tried pET41b+ as it offers more direct expression with an inbuilt c term his tag. or you can try pET47 which has an n term his tag with a 3c protease site for removal.
i normally use pGEX-6P-1 for GST fused proteins and it works well even with the new expression system=arcticexpress de3




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