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SPR identification of mild elution conditions for affinity purification of E6 oncoprotein, using a multivariate experimental design
Author(s) -
Sibler AnniePaule,
Baltzinger Mireille,
Choulier Laurence,
Desplancq Dominique,
Altschuh Danièle
Publication year - 2008
Publication title -
journal of molecular recognition
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.401
H-Index - 79
eISSN - 1099-1352
pISSN - 0952-3499
DOI - 10.1002/jmr.865
Subject(s) - chemistry , elution , surface plasmon resonance , dissociation (chemistry) , affinity chromatography , peptide , chromatography , kinetics , quantitative structure–activity relationship , biochemistry , stereochemistry , nanoparticle , enzyme , materials science , physics , quantum mechanics , nanotechnology
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 ≥ 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°C) were adequate for purifying the recombinant E6 protein from crude cell extracts. Copyright © 2007 John Wiley & Sons, Ltd.