SPR identification of mild elution conditions for affinity purification of E6 oncoprotein, using a multivariate experimental design.
The purification of "difficult" proteins for structural and functional studies remains a challenge. A widely used approach is their production as fusions with an affinity tag, so that a generic tag-based purification protocol can be applied. Alternatively, immuno-affinity using a protein-specific antibody allows purification of unmodified proteins in a single step, if mild elution conditions can be identified for dissociating the complex without disrupting the folding of the protein. Here, we describe a quantitative structure activity relationship (QSAR) strategy to predict optimized elution conditions from a mathematical model that relates target/antibody dissociation to environmental changes. We illustrate the strategy with the E6 protein of the human papilloma virus (HPV) 16, a highly unstable protein central to HPV-induced carcinogenesis. Surface plasmon resonance (SPR) was used to measure the kinetics of dissociation of an E6 peptide from an E6-specific antibody in a set of multivariate conditions, where three environmental factors (pH, NaCl concentration, and temperature) were varied. The QSAR model indicated that dissociation is favored at pH < 5, which is detrimental to E6 folding, and also at pH > or = 10 if the temperature is high. We verified that the conclusions of the QSAR study with the peptide were valid for the scFv1F4/E6 protein complex, and that the recovered protein was capable of mediating p53 degradation. Finally, we demonstrated that the optimized elution conditions (pH 10, 35 degrees C) were adequate for purifying the recombinant E6 protein from crude cell extracts.
University Strasbourg 1, CNRS - UMR 7175, ESBS, Bld Sébastien Brant, BP 10413, 67412 Illkirch Cedex, France.
Annie-Paule
AP
Sibler
Mireille
M
Baltzinger
Laurence
L
Choulier
Dominique
D
Desplancq
Danièle
D
Altschuh
D017033:Anabaena; D000911:Antibodies, Monoclonal / Q000378:metabolism; D000956:Antigens, Viral / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods*; D006801:Humans; D007700:Kinetics; D015999:Multivariate Analysis; D009856:Oncogene Proteins, Viral / Q000302:isolation & purification* / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D021281:Quantitative Structure-Activity Relationship; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D020349:Surface Plasmon Resonance
D000911:Antibodies, Monoclonal; D000956:Antigens, Viral; D009856:Oncogene Proteins, Viral; D010455:Peptides; D011994:Recombinant Proteins