Abstract
BackgroundBacterial expression and purification of recombinant proteins under homogeneous active form is often challenging. Fusion to highly soluble carrier proteins such as Maltose Binding Protein (MBP) often improves their folding and solubility, but self-association may still occur. For instance, HPV E6 oncoproteins, when produced as MBP-E6 fusions, are expressed as mixtures of biologically inactive oligomers and active monomers. While a protocol was previously developed to isolate MBP-E6 monomers for structural studies, it allows the purification of only one MBP-E6 construct at the time. Here, we explored a parallelizable strategy more adapted for biophysical assays aiming at comparing different E6 proteins.ResultsIn this study, we took advantage of the distinct size and diffusion properties of MBP-E6 monomers and oligomers to separate these two species using a rapid batch preparation protocol on affinity resins. We optimized resin reticulation, contact time and elution method in order to maximize the proportion of monomeric MBP-E6 in the final sample. Analytical size-exclusion chromatography was used to quantify the different protein species after purification. Thus, we developed a rapid, single-step protocol for the parallel purification of highly monomeric MBP-E6 samples. MBP-fused HPV16 E6 samples obtained by this approach were validated by testing the binding to their prototypical peptide targets (the LXXLL motif from ubiquitine ligase E6AP) by BIAcore-SPR assay.ConclusionsWe have designed a rapid single-step batch affinity purification approach to isolate biologically active monomers of MBP-fused E6 proteins. This protocol should be generalizable to isolate the monomer (or the minimal biologically active oligomer) of other proteins prone to self-association.
Highlights
Bacterial expression and purification of recombinant proteins under homogeneous active form is often challenging
HPV16 E6 F47R 4C/4S is a solubility-optimized mutant of HPV16 E6. The structure of this mutant has been solved by X-ray crystallography and several of its interactions have been precisely characterized by surface plasmon resonance (SPR) and Isothermal Titration Calorimetry [22]
We investigated the purification of two human papillomaviruses (HPV) E6 oncoproteins fused to Maltose Binding Protein (MBP): the solubility-enhanced mutant HPV16 E6 F47R 4C/4S (16E6mut) whose structure was solved by X-ray crystallography, and the HPV8 E6 (8E6) which, in our experience, was recalcitrant to purify as a monomer
Summary
Bacterial expression and purification of recombinant proteins under homogeneous active form is often challenging. Under the stress induced by high rates of heterologous protein expression, inactive misfolded polypeptides accumulate as inclusion bodies They can be solubilized by using chaotropic agents (urea or guanidium hydrochloride) and refolded in vitro (by dilution or dialysis). An MBP-fused protein can sometimes be solubilized in the form of a mixture of properly folded monomers (or minimal biologically active oligomers) and of large oligomers in which the passenger protein is self-associated and its folding and/or biochemical activity may be altered [5,6,7,8,9,10,11,12,13] In such situations, the challenge consists in exploring conditions of expression and purification favoring the biologically active monomeric -or minimally oligomericMBP-fused samples [9, 11, 14, 15]
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