Hello,
I am trying to crystallize a 16kDa protein. I was unable to express the protein in mammalian system but was successful in expressing it in BL21 (DE3) cells with the addition of a GST tag. Without the GST tag, the protein is insoluble. I have tried different bacterial vectors with His tag and MBP tag but was unable to express the protein.
Since without the GST the protein is insoluble and I do not have any other sources to express the protein, is it possible to crystallize the protein along with the GST tag? Will the GST affect the overall structure of the protein? I do not know if anybody has done it before.
Any help or suggestions will be fantastic.
Thank you for your time and help.
Sayan
Hi
You should use linker between your protein and GST protein. It might help.
Good Luck !!
Dear Sayan
in principle you can certainly solve the structure of the entire GST-protein fusion. The problem is that with out GST the protein is insoluble is it possible that it is intrinsically unfolded and it remain soluble just thanks the solubility of GST and probably you will not be able obtain crystals (due to disorded caused by the unfolding) or in case you will obtain the structure of an aggregate form.
Did you tried to remove the GST tag with TEV or other enzime? Your protein precipitate after GST removal or it remain soluble??
I tried cleaving the GST off by TEV protease but without the GST , the protein aggregates.
Dear Sayan,
you don't give much detail in your protein (for instance, why start with a mammalian system and not bacterial?), but here's my two cents:
1. have you looked at predicted transmembrane domains?
2. does your protein needs any PTMs?
3. have you searched for similar proteins in the PDB? Does this gives you any idea on the fold and/or disordered domains?
4. if no structure available, have you looked at predicted disordered loops/domains?
5. does your protein needs any partner/chaperone? If so, try duet expression.
If you want, we can discuss off line.
Best regards,
Jose
Hi Sayan,
I wonder if you have solved the problem you encountered in the experiment. I have the same question as Jose, which is why start with a mammalian system and not bacterial? In my experience, I will try various construction and expression conditions in the E. coli expression system. If these don’t work, I will choose to try insect cells, such as Sf9, before mammalian cells. Moreover, in theory, the mammalian expression system is very good, and proteins that I have previously found difficult to express or purify in the E. coli expression system have performed well in the mammalian expression system.
Then, your protein is smaller than the GST tag, and there is a linker between GST and the protein. If you take this fusion protein directly to crystallize, you’re likely to get only GST crystals, because GST is very easy to crystallize.
Good luck!
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