I have a protein of 5.6 kda that was contaminated by its pro form (having 4 amino acids MKNM extra). Can I separate them using any type of chromatography? PI of both proteins are 6.7 and 6.9 (theoretical).
I wouldn't expect 'traditional' chromatography (IEX, etc.) to work due to the limited difference between both (unless you would have a specific antibody). Did you try gel filtration? If yes, which column?
If this doen't work, I would either try to avoid this problem (why did it happen anyway?) or if unavoidable, maybe try RP-HPLC or IEX-HPLC.
Thanks for input,
Silver, I tried S100 HR 16/60 column of GE healthcare as well as also tried on KW803 HPLC gel filtration column. for IEX I am using Capto S resin of GE with acetate buffer ph4.8.
I don't think I can avoid this problem, becoz it was a natural protein produced by bacterial strain (not recombinant).
I tried on RPHPLC but still i did not able, may be I am not using right solvent system.
Do u have any suggestion..... please
If it is a bacterial pro-protein the N-terminal methionine should be formylated. You could try to see if anti formyl methionine or formyl peptide polyclonals bind the pro form.
Since you introduce two extra Met residue in your peptide you can easily induce an hydrophilic shift of several minutes on RP HPLC column if you convert by mild oxidation your two Met residues in sulfoxide (SO). This should allow you to separate the two forms
Dear Pardip
If you bacterial peptides have an amphipathic character (antimicrobial peptides perhaps?) separation with C18-HPLC is quite possible. This size of peptide can generally handle harsh treatments and we were able to separate such peptides with one amino acid difference on HPLC. So I would suggest the following as a start for your HPLC separation: (1) C18 matrix - I prefer 150 mm x 39 mm C18 Novapak from Waters for analytical HPLC of peptides; (2) Solvent A - 0.1% TFA; Solvent B - 90% acetonitrile:10% A (do not add extra TFA); flow rate 1 mL/min. If the peptides are amphipathic you can try the following program: equilibrate 5 min with 10%B then inject; 1 min 10% B, 2-15 min gradient from 10% to 100% B; 2 min 100% B column wash; recondition reverse gradient 3 min 100% to 10%B, then back to 5 min equilibration before injection. This is a short 25 run which you can adapt by lowering the starting %B if you peptides elute too early, and incrementally increase the %B if they elute too late – the peptides must ideally elute between 4 and 12 minutes. You can also change the gradient from linear to concave or hyperbolic and see how that improves the separation, as well as increase the temperature to 30, 35 or 40oC. If need be you can increase the runtime by running the gradient from 2-20 min. Once you have optimised your separation you can transfer the method to the same semi-preparative C18 Novapak column by just increasing the flow to 3 mL/min. You also do not need 100% resolution of your two peptides. We found that even if you get peak broadening you can collect the start and tail of the peak separately to obtain pure samples for further analyses.
Hope it helps.
Regards
Marina
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