Hi Maria, As mentioned by Tony, it could be a chaperonin. Having little of ATP (0.5mM or less) and Mg2+ (1mM) in lysis buffer might help.
Good Luck, Partha 2012/3/22 SANCHEZ BARRENA, MARIA JOSE <[email protected]> > Dear all, > > I am trying to express a eukatiotic protein (E. coli codon optimized > sequence) with a GST tag at the N-terminus. I always get my overexpressed > protein and a contaminant around 60kDa. This contaminant is not washed out > of the column when washing glutathione beads with 1M NaCl-buffer. However, > during o/n incubation with proteases that cleave the GST off (thrombin or > TEV), the contaminant is in the soluble fraction..... > > Has someone had this experience? I know about contaminants that bind to > Ni2+ when overexpressing a His-tagged protein, but this is the first time I > get such thing with a GST-tagged protein. > > One could think that that "contaminant" could be a protein that binds to > my overexpressed protein, but I do not think so, cause I always get a huge > band of the contaminant, independently on the amount of the protein of > interest.... > Many thanks in advance for all your suggestions and sorry for asking about > non-crystallographic topics. > Regards, > > Maria > > > ---------------------** > > María José Sánchez-Barrena, PhD** > > Departamento de Cristalografía y Biología Estructural.** > > Instituto de Química Física Rocasolano. CSIC** > > Serrano 119. 28006 Madrid (Spain)** > >
