Hi!
I have tried to expressed my recombinant protein with and without GST.
Without GST, low expression and cannot be purified; with GST, I could not get a clean separation and it did not help solubilized the protein.
I am thinking to use SUMO instead of GST. I have read a few papers showing SUMO can increase the expression and solubility. But I could not figure out what makes it better than GST.
Thanks for answering!
Regards,
Wan Ling
well, Both of them can help for protein solubility and folding while expression. But GST forms a dimer. Sumo tag can be cut off with sumoprotease left a seemless target protein. Some proteins work better with one than another. Just need to test.
Hi,
many 'solubilizing' fusions like GST, MBP etc. will in some cases produce soluble protein that is not necessarily well-folded, so after cleavage your protein may still precipitate. In my experience more proteins stay soluble after cleavage from fusions like Trx and SUMO than with e.g. GST and MBP, that suggests to me that these fusions are actually helping our proteins re-fold.
Another advantage of the SUMO system is the specificity of the protease and the lack of amino acids left on the n-term after cleavage so it is very useful for peptides etc. as well as larger proteins where you want to define your n-terminus.
As Zigou has already pointed out GST dimerizes which may be a problem for some applications (binding constants etc.) but may still work for your protein.
Sometimes you may have to try more than one fusion and it can be useful to find a suite of vectors with the same cloning sites etc. so this can be done in parallel with the same PCR/RE fragment.
The effect of protein tags on expression and solubility are really protein-dependent, so you might want to try several fusion proteins to find one that works for your proteins. In my experience, GST is actually one of the worst for solubility, because its intrinsic dimerization propensity.
- Badges
- Science method
- Similar topics
- Biological Science
- Genetics
- Gene Expression
- Protein Expression