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Only GST alone seems to express


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#1 kos002

kos002

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Posted 07 December 2009 - 10:55 AM

Hi all,

I am currently trying to express 6 GST-fusion constructs using BL21 pLysS cells. All constructs have been verified by sequencing the plasmids and then transformed into the expression cells. To maximize the chance of "seeing" my expressed constructs, I did a "quick GST purification", basically bound them onto the beads, washed 3-4 times and then run a few beads on an SDS PAGE gel. I can see over expression of one of my constructs but with the other two, I get a massive band at about 25-27 kDa which is the size of GST alone! How can this be? I read in a discussion in this forum that cleavage maybe an issue especially if constructs have a thrombin site. Interestingly, the three that show the very strong band around the molecular weight of GST have a thrombin site.... If however this is the problem then should I not get a band at a mass which would be the sum of GST + until the point where thrombin cleaves the protein? For two of the three, thrombin cleaves at around amino acid 100 which then if you add the molecular mass of this "piece of protein" should 26 kDa (for GST) + 10 kDa(up until the thrombin cleavage site) so I should get a distinct band at around 36 kDa? Am I thinking about this the right way?

Any suggestions to overcome this problem? I am expressing in LB. I grow cells at 37 degrees to an OD of around 0.8 then induce with 0.2 mM IPTG at 37 degrees for about 4 hourse before I harvest the cells. My lysis buffer is PBS +150 mM NaCl + 1% Triton + a tablet of complete protease inhibitors from Roche.


Many thanks in advance for your help
Kos002




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