Hi there,

One cant't anticipate the behavior of a protein in a buffer just by analyzing its primary sequence. Tertiary sequence (3D structure) would tell much more about what are the surface residues and therefore what would be the best condition to keep it soluble and folded. But there is nothing better than experiments to confirm.

Hi,

I would suggest using Protparam tool for new proteins. It, at least, can suggest which pH is suitable for your protein and which ion-exchangers can be used for purification. In your case (pI 4.68) the protein should be stable at pH equal or higher than 6 (I would go for 7 or 7.5). Under this conditions, it is negatively charged and should bind to anion exchangers, like Q-sepharose.

Also, blast tells me that it's alpha-lactalbumin, so one can include some calcium for stability. Alpha-lactalbumis are known for their S-S bonds, so I wouldn't be surprised that its purification is problematic due to the chaotic intermolecular S-S bonding, which often leads to precipitation. It might be necessary, therefore, to denature and refold it in the presence of stabilizing agents, like the mix of oxidized and reduced glutathione.

Hope this helps a bit, but I suspect that there is some literature on it with some empirical data. However, sequence alone can tell a lot :)

Good luck!

DEar Xavier

considering the pI= 4.6 theoretically a pH=7 buffer as PBS should be ok

However i have 2 main concerns:

Analyzing the protein sequence with

TMpred Server (vital-it.ch)

SignalP-5.0 (dtu.dk)

1) The protein at the N-terminal contain a signal peptide which is removed when expressed in vivo. How did you express the protein? In mammalian cells or in E.coli?

Because if you express it in a different host is it possible that the signal peptide will be not removed and your protein will contain this N-terminal hydrophobic region will may make it prone to aggregate and poorly soluble in water buffers.

2) The protein contain 8 cysteines and i suspect that at least some of those are invoovled in S-S bonds. Mammalian cells (eg Expi293) are able to form S-S bonds while this not happen efficienty in bacteria expecially if you are performing citoplasmic expression

In this case is important to undertands if the S-S bond are correctly formed because if not, your protein will aggregate. You can stop the aggregation adding on yuor buffer a reducing agent as DTT or TCEP but you will unfold the protein. If you S-S bond are correctly folded you do not need any reducing agent.

I know that i provide you more doubts that solutions but the details are very important with protein .

Ciao

Manuele

This is dog alpha-lactalbumin precursor.... If you produce it in the right system, in a way that it gets secreted, the product will surely be soluble in water. How can the major protein component of milk not be soluble? Perhaps Ca2+ would be a good idea to include, since it is Ca-binding protein. But it is not folded, it will not be soluble (in anything other than 8M GuHCl).