Stability of GST-Fusionproteins with Precission-site - (Oct/19/2007 )
Hi have a strange problem with a GST-fusion protein.
I have cloned a protein (native about 30 kDa) into pGEX6P (with precission protease cleavage site between GST and my protein). My protein lacks its endogenous ATG and is cloned in frame via the BamHI site of the vector directly after the protease site giving the minimal number of additional AA (Leu-Gly-Ser) between the protease site and my protein. The sequence is confirmed.
Nevertheless when expressing it in BL21 I can detect a very prominent band of GST at ~26 kDa in the lysate (soluble fraction) with Coomassie as well as in a western with a polyclonal anti GST. Unfortunatelly there is no antibody agains my protein.
I do not see a visible overexpression band of my protein at its native size in a coomassie.
So what happend? I thought that E.coli endogenously cannot cleave precission-sites.
It is also pretty unlikely that te next ATG after the deleted one from my proetin was used as this would be about 300 bp later and (as it is still in the normal reading frame of the protein) would result in an active protein which i do not see in an activty assay of the lysate.
Surprisingly I can detect the GST-fusion proetin in the insoluble fraction but in the soluble fraction it´s only the pure GST.
Has anybody any idea, where the fusionprotein ended up?
Without knowing more about your data, it seems most likely that some of your cells were transfected with "empty" pGEX that did not contain your fusion protein. I am inclined to think this because GST-fusion proteins are extremely stable.
One possibility that is very easy to undertake is simply purifying your plasmid by agarose gel electrophoresis. Purify your plasmids with a Qiagen midiprep kit or equivalent, from large preps (250mL LAMP o/n culture). Then linearize the plasmid and load this into a preparatory agarose gel (easy if you have a 2-well comb). In a perfect world, you would get two bands: one pGEX without your gene; and one pGEX with it. Simply use a Qiagen gel extraction kit to extract the desired band, re-ligate your plasmid, and transform fresh BL21 cells with this plasmid. Provided you cut the band out precisely enough, this will eliminate "empty" pGEX vector.
Best of Luck!