CABELL.ANDREWS@BIONET-20.BIO.NET (Paul Andrews) (06/15/89)
Protein Isolation:
I am interested in isolating an inner mitichondrial membrane
protein which has been expressed as a fusion protein in an E. coli strain.
Unfortunately, becasue of its extreme lipophilicity, the only way I have been
able to pull it out from the bacteria is with hot SDS resulting in extensive
denaturation of the protein. Anyone have experience in isolating these
type of membrane proteins in an active form from this type of matrix?
--------------------------------------------------------------------------
Paul Andrews ARPANET: CABELL.ANDREWS@BIONET-20.BIO.NET
Univ. of Texas at Austin Phone 512-471-1731
College of Pharmacy
-------MSZ@bmc.uu.se (Michael Szardenings) (06/16/89)
In article <12502328219.30.CABELL.ANDREWS@BIONET-20.BIO.NET>, CABELL.ANDREWS@BIONET-20.BIO.NET (Paul Andrews) writes: > Protein Isolation: > I am interested in isolating an inner mitichondrial membrane > protein which has been expressed as a fusion protein in an E. coli strain. > Unfortunately, becasue of its extreme lipophilicity, the only way I have been > able to pull it out from the bacteria is with hot SDS resulting in extensive > denaturation of the protein. I have only one suggestion : During the expression of an extremly hydrophobic variant of a protein to be secreted by E.coli, I recognized that E.coli may even grow in media containing 1% Triton X-100 (JM103). It increased the yield of protein up to 200%. If Triton or related detergents do not interfere badly with your protein try this. Perhaps that may reduce initial membrane association of hydrophobic stuff and keeps a certain amount soluble. Good luck Michael