I have expressed my recombinant protein using E.coli and refolded it with dialysis method after inclusion bodies isolation and HPLC purification.
I have also checked the mass of the the protein using mass spectrometry and the mass is within my expectations. (with refolded protein - 8 of the actual protein mass due to the formation of 4 disulfide bonds)
my proteins sequence is similar to the actual protein isolated from venom with an addition methionine at the N terminus.
I have no idea why is my protein not inhibiting the target enzyme. Any help or advice is appreciated. Thank you.
Your protein may have a fold, but not the proper folding required.
Are the disulfide bonds correctly formed; i.e., have the thiols that should react with each other reacted or was there a mix-up?
How did you confirm the formation of disulfides?
Is the N-terminus involved in the inhibiting activity? Does the n-terminal met screw things up?
Or, is the protein in its natural host post-translationally modified?
Does the protein act alone or are there other factors involved in the activity, like metal ions?
You say you refolded your protein. Did you use urea? If, yes, did you avoid conditions that lead to carbamylation?
Hi there,
Did you pass your refolded fraction onto a SEC system to check the quality?
Achim Recktenwald Thank you for your reply. I purify the inclusion bodies extract using RP-HPLC. Then I confirm its identity using mass spectrometry by checking its mass (unfolded form). Inclusion bodies extract is prepared by a series of sonication, wash with Triton X-100 buffer and finally with GnHCL buffer to denature it.
Regarding your first post, I confirm the formation of disulfide bonds using mass spectrometry. Ideally, there should be a reduction of 8 da in mass due to the lost of 8 hydrogen atom from the formation of 4 disulfide bonds. However, I was not able to confirm if the desired disulfide bonds were formed in the correct manner.
The actual protein is unlikely to be post-translationally modified in its host as the mass of the actual venom protein is assessed in previous studies using mass spectrometry and there is no abnormal increase in mass. The protein is also not known to have a cofactor.
I am not sure if methionine could be the reason, but my supervisors believed that it is unlikely for a single amino acid to affect the function and structure of the protein.
I did not use urea for refolding. Instead I refolded my protein using denaturing buffer (3M GnHCL, 10 mM EDTA, 20 mM Tris-Cl at pH 8.0) and refolding buffer ( 0.425M GnHCL, 20 mM NaCl, 1mM reduced glutathione, 1mM Oxidized glutathione, 1mM EDTA, 50 mM Tris-Cl, 10% glycerol at pH 8.0.
Im not sure what else can I do to improve my refolding. I also tried refolding using iodine oxidation method (add iodine, purge with N2 gas and quench using ascorbic acid) but it still didnt work.
Dominique Liger Thank you for your reply. No I didn't. I pass my refolded fraction through the RP-HPLC again and assessed its mass with mass spectrometry. By SEC system, are you referring to size exclusion chromotography ? How does it check the quality ? Sorry, i'm new to this.
Hi again, SEC is very convenient to check if your protein is agregated or not. If it is properly folded it will be eluted according to its apparent MW (which may vary depending on the oligomerization state). If not it will be eluted much earlier.
Dominique Liger hi, shouldn't the mass spectrometry be able to tell us that ?
I am thinking if my protein is aggregated, the mass detected would be higher than the expected size.
Hi again, MS is a denaturing process therefore it can't be used for non covalent assembly such as protein oligomers or agregates.
The met at the N-terminus could interfere, if the N-terminus is part of the binding site or is close to the binding site in the properly folded protein.
One thing you could try is play with your refolding conditions. 3M guanidine is at the low end for unfolding a protein. You may only have unfolded it partially in 3M. I would try 6M and then in steps down to 0M. Also, you use reduced/oxidized glutathione in a 1:1 ratio. This is at the high end for ox. glutathione. I would play with this number; I usually used 3:1, 5:1, or even 10:1.
Achim Recktenwald Dominique Liger Thank you so much for your suggestions. I will try it out in the lab.
I can imagine that the cysteins in your protein are not forming the native combination of disulfides betwen each other. In your position I would try this system: Article Efficient soluble expression of disulfide bonded proteins in...
Together with MBP protein and special disulfides tolerant TEV cleavage protocol.