SPR identification of mild elution conditions for affinity purification of E6 oncoprotein, using a multivariate experimental design

Abstract

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.

Development of an aptamer-based affinity purification method for vascular endothelial growth factor

“…The ions may interfere with either intermolecular electrostatic interactions between aptamer and protein or with intramolecular interactions between the aptamer’s nucleobases resulting in destabilization of aptamer folding. Salt-dependent protein elution is a mild elution strategy which may avoid protein denaturation [23] . For all aptamer variants it was possible to elute at least 75% of the bound protein which is a promising basis for VEGF purification.…”

Spot peptide arrays and SPR measurements: throughput and quantification in antibody selectivity studies

“…Both buffers were filtered through a 0.22 µm membrane and supplemented with 0.005% (v/v) surfactant P20. The WT decapeptide of sequence 6 TAMFQDPQER 15 and its variants were synthesized with a C‐terminal cysteine and were covalently coupled on sCM5 surfaces using the thiol coupling chemistry as previously described (Sibler et al ., ) to reach maximal scFv1F4 binding capacities in the range 10 and 300 RU. The reference surface was treated as the peptide surfaces except that peptide injection was omitted.…”

“…ScFv1F4-B10 and scFv1F4-Q L34 S-B10 (scfv1F4 with a Q34S replacement in the light chain; Hugo et al, 2002) cloned in pscFew vector (Giovane et al, 1999) were expressed in E. coli BMH 71-18 cells. They were purified from crude periplasmic fraction by affinity chromatography on Sepharose 4B coupled either with Nter_MBP-E6-peptide 1-18 as previously described (Sibler et al, 1999(Sibler et al, , 2008Hugo et al, 2002) or with the synthetic peptide 4 KRTAMFQDPQERPR 17 .…”