I'm trying to express an antibody fragment that is covalently held together by cysteine residues that I have added to the sequence. After running the western (non-reduced) to assess expression, I obtain two bands. One band represents half of the fragment, the other is the covalent version at double the size (cys-fragment). I'd like to increase the amount of covalently bound fragment.
Does anyone have experience with the addition of copper sulfate to the media to promote thiol bonding? I've found some literature from Chaderjian et al, which was helpful, but I'm also curious if the copper will inhibit purification via 6-his tag?
copper binds very strongly to His-tags, so you would have to first wash your cell lysate in EDTA, and then wash EDTA very carefully and very efficiently away before loading on a Ni or Co column. Otherwise maybe try to precipitate the copper with for example phosphate? And imidazole to fasten the process?
As I understood you wanted to get a dimer therefore I would try to get the dimer at the conditions that facilitate the dimer creation, another words you should try to do the opposite way that is usually done to prevent the dimer creation, and one of them is high concentration of protein if you keep it in solution with relatively high concentration of reduced agents at pH arpund pH=8.0 and higher.
Adding Cu to the growth media will not likely have much effect on expression of a protein inside the cell. Most cells maintain very low levels of intracellular copper regardless of the concentration in the media. I have expressed a copper-containing protein in the periplasm of E. coli and added copper to the media which helped its expression. But in that case the periplasmic space tends to equilibrate more with the media than the cytoplasm. Also, the copper in the growth media should not interfere with his-tag purification. The media is removed before prepping the cells and the concentration in the lysate should be minimal.
Thank you all for the helpful responses. I am expressing the protein in 293F mammalian cells, so I'm more concerned about maintaining cell viability after addition of copper. Also my protein is secreted into the media, so no cell lysis. I believe the benefits of adding copper to induce disulfide formation may outweigh the labor intensiveness of trying to remove the copper from the media prior to his purification.
Hello Jason
I am currently working on a research project involving addition of copper. There are two things I'd like to add to the answers above.
First, in mammalian culture, addition of copper can actually improve production and increase viability throughout your culture. If you are using a chemically defined media without trace elements, it's highly probable that the cells are copper deficient. With addition of copper, I would assume you would see increased culture efficiency. If you are/did see a drop in viability upon addition, this may be due to the fact there are not enough chelating thiols to facilitate uptake and protect the cell from coppers redox potential. I would recommend adding copper to the media prior to exposure to cells to allow the DO and chemistry to equilibrate.
Second, as you read in the paper from Chaderjian, the results from addition to copper were decreased free thiols. This may not be beneficial to you. I understand your protein is held together by cysteine residues, but Janice Bramham was correct above when saying you do not want a reducing environment. The copper will not only catalyze the reaction in formation of your protein but will also catalyze the formation of disulfide bonds between any available thiols. You may have seen increased aggregates that would have been less likely without copper.
It ties back to the first paragraph. I recommend adding copper pre-inoculation with plently of free thiols to chelate. This will facilitate uptake and utilizaiton in protein production and cellular metabolism. I believe excessive copper in your media will increase the amounts of unwanted aggregates.
@Matthew - I gave your suggestions some thought, and what about adding copper to the media after harvesting it from production? Since in my case the protein is secreted into the supernatant, the cells have long been filtered away prior to purifying on the Ni-NTA column. At this point I don't need to worry about cell viability because I'm only concerned with the supernatant. Considering I am am using serum-free media during production, there might be a benefit to adding copper to induce disulfide formation once the supernatant is harvested. The copper could then be removed by dialysis prior to purifying the supernatant on an NI-NTA column. I could be way out in left field though, as I have no experience with this.
Sorry for such a late response
I didn't understand originally that you wanted to induce di-sulfur bond formation after excretion. I've found quite a few papers on copper catalyzed thiol chemistry in the past that should be helpful.
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