Hi, I am expressing a MBP-fusion protein with 4 cysteine and I see free cysteine on LCMS indicating the disulfide bond formation is not complete while expression. Is there any way to facilitate the the formation of S-S bond except the air-flush and refolding process.
From your question I deduce that you produce your protein by cytoplasmic expression in E-coli and refold it from inclusion bodies - if I am wrong, give more details of what you are doing.
Air-oxidation can be catalysed by the presence of trace amounts of copper ions:
"The formation of disulfide bonds was catalyzed either by the presence of reduced and oxidized glutathione in the refolding buffer (standard molar ratio of GSH to GSSG was 0.2:1.0, but other ratios were tested), or simply by air oxidation, catalyzed by 5 microMolar CuSO4 ."
From: https://plueckthun.bioc.uzh.ch/wp-content/uploads/Publications/APpub0114.pdf
Alternatively, you can try either periplasmic expression or the use of an e-coli strain engineered to provide a more oxidising environment in its cytoplasm.
Article Production of disulfide-bonded proteins in Escherichia coli
Deay Yuhan
if you produce the protein in E.coli cytoplasm is quite normal that your disulfide are not well formed because E.coli cytoplasm is a reducing environment.
You can try to:
- Express the protein in Origami E:coli strain
- Add to the protein a N-terminal leader peptide and express it into the periplasm which is more oxidizing and permit the formation of S-S bonds.
You can also try to facilitate S-S formation in vitro after purification by adding different ratio of GSH/GSSG or but there is some risk of aspecific intramolecular S-S bond formation and protein aggregation.
During my phD i find out that a mitochondrial protein Mia 40 was able to catalyse in vitro che oxidation of S-S of proteins containing CX9C or CX3C motif
https://www.nature.com/articles/nsmb.1553
but i did not have the opportunity to check if in vitro in can improve the oxidation also of S-S from other proteins.
good luck
Manuele
I would recommend looking into CyDisCo expression system.
The CyDisCo is an plasmid that allows any Ecoli strain to support proper formation of disulfide bonds at any media.
You can read up more about it here:
1) https://doi.org/10.1186/s12934-017-0721-x
2) https://www.oulu.fi/sites/default/files/content/files/Technology%20Offer_CyDisCo_UnivOulu.pdf
Yuhan Lyu , thank you for the feedback - I am glad this worked for you.