Cleavage of target protein from glutathione sepharose - (Jul/25/2006 )
Hello all I'm a newbie here so please bear with me.
I am working with a VERY troublesome protein. First I had a problem of expression in bacteria (protein going into inclusion bodies), which was solved by expressing my GST fusion protein in Stratagene's Artic Express cells. Now I am having a problem in cleaving my protein from GST using both Thrombin and Precission Proteases. Ok, maybe the problem isn't necessarily the cleavage but liberating the "cleaved" protein from the glutathonine sepharose. I have tried various elutions methods like using various concentrations of NaCl and Triton-X; 1M and 1% being the highest concentration used, respectively. Also, I've used various elution times with 24 hours being the longest. As a final straw, we have even used 1% SDS and boiling the sepharose to liberate my cleaved protein. After many comassie and silver stained gels later, it appears that the cleavage works because there's a correctly sized band for my protein; but most of the protein remains in the sepharose (I would say that I elute at the most about 5-10% of my protein). I have even tried to do the cleavage step on the bacterial lysate prior to incubation with the glutathione, with the same results. Oh the lab uses the batch purification glutathonine sepharose from Amersham, GE life, or whatever the hell they go by nowadays. Would it be better if I used the column system, which my PI thinks is worse?
Please help my antibody development and ultimately my PhD depends on this.
The GST protein is generally eluted by reduced glutathione, have you tried to elute the protein before cleavage? Sometimes the binding of protein with resin blocks the cleavage site so that it can't be easily cleaved out directly.
I am working with a VERY troublesome protein. First I had a problem of expression in bacteria (protein going into inclusion bodies), which was solved by expressing my GST fusion protein in Stratagene's Artic Express cells. Now I am having a problem in cleaving my protein from GST using both Thrombin and Precission Proteases. Ok, maybe the problem isn't necessarily the cleavage but liberating the "cleaved" protein from the glutathonine sepharose. I have tried various elutions methods like using various concentrations of NaCl and Triton-X; 1M and 1% being the highest concentration used, respectively. Also, I've used various elution times with 24 hours being the longest. As a final straw, we have even used 1% SDS and boiling the sepharose to liberate my cleaved protein. After many comassie and silver stained gels later, it appears that the cleavage works because there's a correctly sized band for my protein; but most of the protein remains in the sepharose (I would say that I elute at the most about 5-10% of my protein). I have even tried to do the cleavage step on the bacterial lysate prior to incubation with the glutathione, with the same results. Oh the lab uses the batch purification glutathonine sepharose from Amersham, GE life, or whatever the hell they go by nowadays. Would it be better if I used the column system, which my PI thinks is worse?
Please help my antibody development and ultimately my PhD depends on this.
I had a similar problem with a construct I had. I was able to elute the sticky protein from the resin using 1% Sarkosyl in the elution buffer and eluting dropwise in column, maybe ypou could try it that way. But I warn you, it´s not so easy to dialyze later on. I suppose that you don´t need completely functional protein to immunize, but anyway, the protein doesn´t remain complete native neither.
Good luck
Is the Sarkosyl stronger than SDS, because I just assumed that SDS would be the harshest detergent used for elution. However, I could be wrong.