Hello,
I am working on a recombinant protein named Brolucizumab. the protein is designed as a monomer with a molecular weight of 26kd. It has 4 cysteines that form 2 s-s bind. I have a problem with this protein when I solubilize and Refold it. I solubilized it with 8M urea, tris50mM, and EDTA 10mM. For reducing the disulfide bond fresh dtt 10mM was used. after the protein was solubilized the solution was transferred to the refolding buffer in a ratio of 1/10. refolding components are L-Arg, Sorbitol, Tris, EDTA, urea 2M, and Cysteine/Cystine. Everything is fine so far. The problem is that when I solubilize Brolucizumab, ،The proteins tend to form dimers and trimers. the problem was solved by adding ddt in solubilization but after refolding you can see dimers and trimers. I monitored all of the steps by sds-page in both forms with and without BME.
at this time I don't have any idea how to solve my problem. can anyone help me with this problem?
Thanks.
You may be able to separate the monomers from the dimers and trimers by gel filtration chromatography.
You could try refolding with different initial protein concentrations. The lower the concentration the lower the number of inter-molecular interactions. Alternatively, you accept the result and separate the dimers and trimers from the monomer.
The name of your protein suggests a full-length antibody, however you seem to have a fragment of it (scFv or processed fragment of the antibody?). Expressed in E. coli? You could try periplasmic expression or Origami strains to get a folded fragment without the need for refolding.
Hi Mostafa, you can prevent intermolecular disulfides (and the formation of di- and tri-mers) by:
1) Using larger volumes of solubilization and refolding buffers (you can start with 1.5x more volume and increase if required). This reduces the concentration of the protein, prevents intermolecular reactions, and increases the yield of the monomer. The problem with this approach - you will have to deal with large volumes, invest more time in concentrating the mixture.
2) Transferring the solubilized protein into the refolding buffer drop-wise, while stirring on ice, over a period of 30 minutes or more. This reduces the concentration of unfolded monomer at any point of time during the process and gives it enough time to fold correctly.
Finally, you must perform SEC (Adam B Shapiro's suggestion) anyway to get rid of higher MW species. Good luck!
Using iodoacetamide in one of the steps to block the disulfide exchange?
Karthik's second suggestion also makes sense.
For this problem, I removed l-arg from my refolding buffer and then l increased sorbitol concentration.
My problem is solved.
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