Kb Homes

Dear all,

I am trying to purify a bacterially expressed peptide using RP-HPLC. In order to use HPLC, I dialyzed the peptide against pure water and lyophilized it. Because it's just pure water with no salt, the peptide precipitates. When I try to solubilize it in water to load on the HPLC, the peptide is still precipitated. I was able to use 50% MeOH to solubilize it for MS. But I fear that MeOH would prevent my peptide from interacting with the RP column. I suppose this happens quite a bit, though I am not sure what people use to overcome the solubility issue? I've read about adding TFA, but only to a 0.07%? I am currently re-expressing the peptide. Would small amount of TFA solubilize the peptide? How much TFA can I add?

Thanks a lot for your time

Hello,

I have a question about peptide expression in e. coli. I am planning on expressing a 16-aa peptide with a 6xHis tag and a Met site in between to cleave the His-tag after purification. So the gene would be: Met-HHHHHH-Met-16aa-STOP downstream of a T7 promoter. I realize most people express small peptides as a fusion with other proteins or larger tags. I've been looking and I can't really find the reason why a small peptide like mine cannot be expressed as is. The peptide itself should be very soluble and the 6xhis tag should help with the solubility as well.
So, the question is, what is the reason for people to use larger fusion partner (eg GST, SUMO)? Is it just due to the small size of the peptide and you want something tangible for gels and columns? or is there any other properties that I'm unaware of such as effects on expression level?

Thank you for your help and input.