Hi all,
I am working on the synthesis of a 21 amino acid residues long peptide with the following sequence- NH2SSSDSSSDSSSDSDSSTDDSY-CONH2. The peptide has mutliple serines and a few aspartate residues. I am using the standard Fmoc coupling chemistry on microwave peptide synthesizer. After running a crude analytical HPLC run, I don't see the mass of the desired product and also nor do I see deletions of any residue. My question is can the synthesis of this peptide be difficult because of its sequence? How should I optimize my peptide synthesis? I was thinking of expressing it as a fusion protein in E.Coli in case my peptide synthesis route fails.
Thanks in advance
Prakriti
Hi Prakriti,
I have also synthesized peptides on microwave peptide synthesizer following SPPS method. If you have a Arg/His in your sequence then some problem arises but other than that there shouldn't be any problem. The sequence is quite simple-
1. Try to attach the first amino acid manually using a pinch of DMAP with all the normal reagents used in SPPS.
2. Your peptide mass should be 2103.8 Da, what are the molecular weight you observing after HPLC?
3. Is their any cyclisation of peptide?
Good luck
Prasenjit
In my experience poly-serine regions are problematic. They are difficult to assemble, because the nascent molecule folds on the solid phase. Problems continue after cleavage, because such peptides are prone to form beta-structure in solution, creating thick gels. Try using a long double-couple cycle for assembly, and then keep pH 6-8 after cleavage.
You should also keep an eye on the resin you're using, polarity and backbone folding are issues that can be solved by choosing the right resin for a particular sequence.
Iris has a good point. And I would add use of a low-substitution resin.
Hello Prasenjit Mondal , thanks for your suggestions. I am seeing some numbers like 1587 and 2232 Da which have no relation with my expected mass.
John Carter
Hello, thanks for your inputs. Yes, I am also seeing gelation of the crude peptide mixture. I have added guanidinium HCl as well to destroy the secondary structure but it didn't have a greater impact. Do you think keeping the peptide in basic pH, will help to keep the aspartate residues in deprotonated form that would aid solubility for HPLC purification?
Another thing that is bugging me is that I am unable to detect the desired product mass in the crude peptide mixture by MALDI as well. I can understand that HPLC runs may be having problem, but crude is also not showing the desired mass. This might mean that my synthesis failed itself.
Currently, I am doing coupling of a small number of amino acids because aspartic acid residues might promote aspartimide formation as well.
pH needs to liberate the aspartate, which converts from weak beta to strong alpha-helix upon deprotonation. :-)
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