A strategy for optimizing the monodispersity of fusion proteins: application to purification of recombinant HPV E6 oncoprotein

Recombinant production of HPV oncoprotein E6 is notoriously difficult. The unfused sequence is produced in inclusion bodies. By contrast, fusions of E 6 to the C-terminus of carrier proteins such as maltose-binding protein or gluthatione-S-transferase are produced soluble. However, it has not yet bee n possible to purify E6 protein from such fusion constructs. Here, we show that this was due to the biophysical heterogeneity of the fusion preparatio ns. We find that soluble MBP-E6 preparations contain two subpopulations, A major fraction is aggregated and contains exclusively misfolded E6 moieties ('soluble inclusion bodies'). A minor fraction is monodisperse and contain s the properly folded E6 moieties, Using monodispersity as a screening crit erion, we optimized the expression conditions, the purification process and the sequence of E6, finally obtaining stable monodisperse MBP-E6 preparati ons. In contrast to aggregated MBP-E6, these preparations yielded fully sol uble E6 after proteolytic removal of MBP, Once purified, these E6 proteins are stable, folded and biologically active. The first biophysical measureme nts on pure E6 were performed. This work shows that solubility is not a suf ficient criterion to check that the passenger protein in a fusion construct is properly folded and active, By contrast, monodispersity appears as a be tter quality criterion. The monodispersity-based strategy presented here co nstitutes a general method to prepare fusion proteins,vith optimized foldin g and biological activity.