Dr Engelbert Buxbaum
engelbert_buxbaum at hotmail.com
Sun Apr 24 07:01:21 EST 2005
> Hi ng
> I have cloned my 2.4bp GOI into pGEX-4T-1 with a N'terminal GST tag and
> expressed the protein in BL21 cells. The problem is that the GST fusion
> protein does not bind glutathionesepharose. By WB using an antiGST abs I can
> see that the protein is expressed at high levels and is soluble!
That can happen, if the affinity site is sterically blocked in the
fusion protein. Two possible solutions:
a) do a conventional purification without using the tag. Given the high
expression of the fusion product, this is usually easy and adequate if
the protein is not required regularly in large quantities.
b) Make a new construct expressing the tag in a different position, or
using a different tag. This is the more permanent solution.
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