4 years ago

Development and Application of Synthetic Affinity Ligands for the Purification of Ferritin-Based Influenza Antigens

Development and Application of Synthetic Affinity Ligands for the Purification of Ferritin-Based Influenza Antigens
Tom Sheppard, Christopher R. Lowe, Nick Bampos, Richard Schwartz, Basmah Khogeer, Shaleem I. Jacob
A recently developed novel recombinant influenza antigen vaccine has shown great success in preclinical studies in ferrets and mice. It provides broader protection, and is efficient to manufacture compared to the conventional trivalent influenza vaccines (TIV). Each strain of the recombinant antigen has a constant self-assembled bacterial ferritin core which, if used as a target for affinity chromatography, could lead to a universal purification method. Ferritin in silico models were used to explore potential target binding sites against ligands synthesized by the four-component Ugi reaction. Two ligands, SJ047 and SJ055, were synthesized in solution, characterized by 1H, 13C, and 2D NMR spectroscopy, and subsequently immobilized on the PEG-functionalized beads. Ligands SJ047 and SJ055 displayed apparent Kd values of 2.04 × 10–7 M and 1.91 × 10–8 M, respectively, against the ferritin. SJ047 and SJ055-functionalized resins were able to purify hemagglutinin (New Caledonia)-ferritin expressed in a crude Human Embryonic Kidney (HEK) cell supernatant in a single step to a purity of 85 ± 0.5% (97 ± 1% yield) and 87.5 ± 0.5% (95.5 ± 1.5% yield), respectively. Additionally, SJ047 and SJ055-functionalized resins purified the recombinant antigens when spiked at known concentrations into HEK supernatants. All three strains, hemagglutinin (New Caledonia)-ferritin, hemagglutinin (California)-ferritin, and hemagglutinin (Singapore)-ferritin were purified, thereby offering an ideal alternate platform for affinity chromatography. Following elution from the affinity adsorbents, absorbance at 350 nm showed that there was no aggregation of the recombinant antigens and dynamic light scattering studies further confirmed the structural integrity of the recombinant antigen. The use of Ugi ligands coupled to a PEG-spacer arm to target the ferritin core of the strain is entirely novel and provides an efficient purification of these recombinant antigens. This approach represents a potentially universal method to purify any ferritin-based vaccine.

Publisher URL: http://dx.doi.org/10.1021/acs.bioconjchem.7b00253

DOI: 10.1021/acs.bioconjchem.7b00253

You might also like
Discover & Discuss Important Research

Keeping up-to-date with research can feel impossible, with papers being published faster than you'll ever be able to read them. That's where Researcher comes in: we're simplifying discovery and making important discussions happen. With over 19,000 sources, including peer-reviewed journals, preprints, blogs, universities, podcasts and Live events across 10 research areas, you'll never miss what's important to you. It's like social media, but better. Oh, and we should mention - it's free.

  • Download from Google Play
  • Download from App Store
  • Download from AppInChina

Researcher displays publicly available abstracts and doesn’t host any full article content. If the content is open access, we will direct clicks from the abstracts to the publisher website and display the PDF copy on our platform. Clicks to view the full text will be directed to the publisher website, where only users with subscriptions or access through their institution are able to view the full article.