Our recombinant protein fused to ABD (albumin binding domain) degraded before and after purification (imax, ABD-agarose) into 2 parts. Protease inhibitors didn’t help. We use linker (G4S)3.
Dear Elena Demyanova
unfortunatelly there is not a simple answer to your question:
Did you express it in E.coli?
Did you already tried to shift down the induction temperature (eg. 17- or 21 and or change the E.coli strain?
Did you know the exact location of you cutting site? (did you performed an N-terminal sequencing of the fragments?) to be sure that the degradation happen at the linker level?
Proteins with unfolded regions or protein fusions with flexible linkers (as G4S)3 is are often susceptible to cutting by proteases of E.coli. However the problem is not ever due to the linkers:
For example in the past i spent long time to try to stablize 2 proteins:
in the fist cases, we worked with a fusion wich contain of 3 different variants of the same antigen (fused to enanche the vaccine coverage) and we observe degradation. in the beginning we were convinced that the problem was in the linker (GSGS) and we spent a lot of effort to replaced it with different linkers (more rigid and with different lenght) but at the ends after N-terminal sequencing with found that the digestion happend inside the N-teminal of 1 of the variants that was characterized from higher and only stabilization by mutagenesis was able to reduce this problem.
In a second case, working of the production of the catalitic domain of clostridium TcdA. A lot of trials were performed in E.coli but in any case insolubility or degradation were observed and we were able to solve it by using a different expression host, the Brevibacillus
Article The structure of Clostridium difficile toxin A glucosyltrans...
good luck
Manuele
- Badges
- Science topic