I have cloned a gene in pGEX-4T-1 expressed in JM109 and purified the recombinant GST-tagged protein using gluthathione sepharose bead column chromatography having confirmed the correct sequence. I then ran samples of the uninduced, induced, pellet, supernatant, flow through and washes samples along with the eluted fractions of my recombinant protein on 10% SDS-PAGE and stained with Coomassie blue. My GST-tagged protein is about 94 kDa. I got a band close to the 100 kDa marker after induction but following lysis and subsequent purification I observed two distinct bands at 100 kDa and at 60 kDa with smaller intermediates. Western blotting with anti-GST primary antibody revealed all to be reactive. Probing with positive sera against the whole cell antigen reveal reaction around 75 kDa only.
The gene also has a leucine rich repeat motif.
You should try using protease deficient expression strains such as BL21. You need to add protease inhibitors, such as Roche c0mplete or Sigma protease inhibitor cocktail. Adding PMSF can also help sensitive proteins. All procedures should be carried out at 4oC to reduce proteolysis. And some proteins are inherently prone to proteolysis.
Is the protein still active? What do you want the protein for? As you are not getting complete degradation, but instead a major band at 60 kDa, this might be the more stable domain, useful for crystallography and some activity assays, after removal of the GST tag. Alternatively you can perform Gel Filtration to remove the 60 kDa protein and just work with the 100 kDa species.
You should try using protease deficient expression strains such as BL21. You need to add protease inhibitors, such as Roche c0mplete or Sigma protease inhibitor cocktail. Adding PMSF can also help sensitive proteins. All procedures should be carried out at 4oC to reduce proteolysis. And some proteins are inherently prone to proteolysis.
Is the protein still active? What do you want the protein for? As you are not getting complete degradation, but instead a major band at 60 kDa, this might be the more stable domain, useful for crystallography and some activity assays, after removal of the GST tag. Alternatively you can perform Gel Filtration to remove the 60 kDa protein and just work with the 100 kDa species.
All procedures were carried out on ice and purification at 4C. I did add lysozyme and PMSF too and incubated at 4 C overnight before sonication on ice. I intend to use the protein for functional analysis.
You definitely need some more protease inhibitors. Also try to carry out your initial purification as quickly as possible to minimize the time for proteolysis. You might like to add a little DNase to help your purification, though it won't help with protein degradation.
Before you embark on this, I would want to know the following:
In vivo or in vitro? Take a cell prep post induction, inactivate all proteases (TCA at 4% will do that), run the pellet on a gel, and do a western. Full size or smaller?
If smaller, then your ectopic proteolysis is in vivo, and you have to ameliorate that by using protease deficient strains, lower temperatures etc.
If full sized, you are much better off, because it is YOU who is causing the proteolysis, and thus, YOU can eliminate it . At this stage, minimise processing time, add a full range of protease inhibitors, and ADD THEM FRESH! PMSF has a half file of about 10minutes in an aqueous buffer, so unless you make dry solvent stocks and use them immediately, it may not be effective. I'd recommend this book "Proteolytic Enzymes", an old book I and Judy Bond edited a long time, and which has strategies for elimination of proteolysis. It's old now, but it is still germane, especially, the concepts.
Systematic, analytical, purposeful. Take your problem apart, one step at a time. Good luck!
BTW, I didn't understand how whole cells gave you 75kDa western band, but the purified protein gave 100kDa. That is the opposite of proteolysis!
I am experiencing a similar problem, using all protease inhibitors (PIC and PMSF) fresh, every step of the purification done fast and on ice (except the binding to Nickel resine for 1 hour), using a sepharose column at 4C for final separation, using rosetta cells that are protease deficient strains. I do not know what I can do to inhibit further protein degradation... Any further suggestion? Adamu, did you make some progress on that already!? I will search for the "proteolytic enzyme" book as well.
Anabel-Lise
you need to establish if the proteolysis is in vivo (before you break cells) or in vitro (after). If in vivo, no inhibition strategy in the tube will help. Please stop expressing cells in 5% TCA and then run a gel on the protein pellet (after acetone washing and redissolution).
Dear Rob, yes you are right, I had seen your comment as well. I will do and let you know, thanks a lot.
I have an additional question no the degradation issue. I actually know that my protein only degrade when it is attached to membranes (via N-myristoylation); if I express and purifiy the non-myristoylated form, it does not degrade... but I just discovered that a purification using thrombin of the non myristoylated protein also degrades the protein. What enzyme could be "thrombin-like" in E.Coli and be close to membrane? Thanks!!!
Dear Adam,
I am recently also experiencing similar issue. My protein reveals a smaller band after SDS-PAGE when left > 3 days at 4 degrees. Ellis commented that BL21 is a protease deficient cells - however I used Rosetta DE3 pLysS; did you happen to use this E.Coli to express your GST protein?
- Badges
- Science method