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)
