I would hazard a guess of Gro-EL. With regards,
Tony. --- Dr Antony W Oliver Senior Research Fellow CR-UK DNA Repair Enzymes Group Genome Damage and Stability Centre Science Park Road University of Sussex Falmer, Brighton, BN1 9RQ email: [email protected]<mailto:[email protected]> tel (office): +44 (0)1273 678349 tel (lab): +44 (0)1273 677512 From: "SANCHEZ BARRENA, MARIA JOSE" <[email protected]<mailto:[email protected]>> Reply-To: "SANCHEZ BARRENA, MARIA JOSE" <[email protected]<mailto:[email protected]>> Date: Thu, 22 Mar 2012 16:40:38 +0100 To: <[email protected]<mailto:[email protected]>> Subject: [ccp4bb] contaminant when overexpressing a GST tagged protein 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)
