One-step affinity purification of fusion proteins with optimal monodispersity and biological activity: application to aggregation-prone HPV E6 proteins

Microb Cell Fact. 2018 Dec 1;17(1):191. doi: 10.1186/s12934-018-1039-z.

Abstract

Background: Bacterial 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.

Results: In 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.

Conclusions: We 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.

Keywords: IMAC; MBP fusion; One-step purification; Protein aggregation; Protein solubility.

MeSH terms

  • Chromatography, Affinity / methods*
  • DNA-Binding Proteins / metabolism*
  • Oncogene Proteins, Viral / metabolism*
  • Recombinant Fusion Proteins / chemistry*
  • Recombinant Proteins / metabolism*

Substances

  • DNA-Binding Proteins
  • E6 protein, Human papillomavirus type 18
  • Oncogene Proteins, Viral
  • Recombinant Fusion Proteins
  • Recombinant Proteins