Hello,
does anyone have a suitable protocol for coupling maleimide-PEG to a protein? The protein has just one cysteine residue (which was genetically engineered), MW is ~ 6.7 kDa, and theoretical pI > 8.
I want to do 2 separate thiol-maleimide coupling reactions:
1) one using a 4-unit PEG of 360 Da (0.36 kDa) and later
2) the second reaction using a 10 kDa PEG
After coupling, purification of the protein / 10kDa PEG conjugate can easily be done by SEC.
But any recommendations on purification strategies for the protein / 0.36 kDa PEG conjugate? Considering that the unbound 0.36 kDa PEG can simply be removed by a spin column, how can I efficiently separate the protein-PEG conjugate from the unconjugated protein? (these two have a mass difference of only about 400 Da). Any columns or resins which can separate these two with a good enough resolution? IEX or SEC?
I look forward to your suggestions.
You can avoid the necessity of separating the PEG4-protein from the unPEGylated protein by using a sufficient excess of PEG4-maleimide in the presence of the reducing agent TCEP that there is not a significant amount of unPEGylated protein left. To see if this is the case, you can try using a reverse phase UPLC or HPLC separation of a C4 column to separate PEG4-protein from unPEGylated protein on an analytical scale.
On a preparative scale, hydrophobic interaction chromatography may be the best approach, using a Phenyl-Sepharose column, for example.
thank you very much Adam B Shapiro. I plan to use 10 - 20x molar excess of the mal-PEG to the protein. On the other hand, I read some papers which suggest TCEP and DTT affect thiol-maleimide reaction, leading to incomplete conjugation of protein. I was wondering, based on your experience, is 10x molar excess of TCEP enough to reduce, but also not have unwanted effects?
I also intend to remove as much of the TCEP by a spin column. But in doing so, doesn't the thiol group re-oxidize? At what steps is it critical to supply inert gas (N2 or Argon)? Or is it even necessary at all to add inert gas to the Eppendorf tubes? Thanks.
TCEP is supposedly compatible with maleimide labeling. DTT, of course, is not. You should use at least a 10-fold molar excess of TCEP. Here is some useful information on the labeling procedure:
https://www.thermofisher.com/us/en/home/references/protocols/cell-and-tissue-analysis/labeling-chemistry-protocols/thiol-reactive-probe-labeling-protocol.html
I don't think flushing the tube with inert gas is necessary.
thanks a lot Adam B Shapiro . I will try this out next week. Keeping my fingers crossed.
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