tetano
Editor, Senior Moderator
Bioconjug Chem. 2017 May 30. doi: 10.1021/acs.bioconjchem.7b00253. [Epub ahead of print]
[h=1]The development and application of synthetic affinity ligands for the purification of ferritin-based influenza antigens.[/h] Jacob SI, Khogeer B, Bampos N, Sheppard TD, Schwartz RM, Lowe CR.
[h=3]Abstract[/h] A recently developed novel recombinant influenza antigen vaccine has shown great success in pre-clinical 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 synthesised by the four-component Ugi reaction. Two ligands, SJ047 and SJ055, were synthesised in solution, characterised by 1H-, 13C- and 2D-NMR spectroscopy, and subsequently immobilised on the PEG-functionalised beads. Ligands SJ047 and SJ055 displayed apparent Kd values of 2.04x10-7 M and 1.91x10-8 M, respectively, against the ferritin. SJ047 and SJ055-functionalised resins were able to purify haemagglutinin (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-functionalised resins purified the recombinant antigens when spiked at known concentrations into HEK supernatants. All three strains, haemagglutinin (New Caledonia)-ferritin, haemagglutinin (California)-ferritin and haemagglutinin (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.
PMID: 28558204 DOI: 10.1021/acs.bioconjchem.7b00253
[h=1]The development and application of synthetic affinity ligands for the purification of ferritin-based influenza antigens.[/h] Jacob SI, Khogeer B, Bampos N, Sheppard TD, Schwartz RM, Lowe CR.
[h=3]Abstract[/h] A recently developed novel recombinant influenza antigen vaccine has shown great success in pre-clinical 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 synthesised by the four-component Ugi reaction. Two ligands, SJ047 and SJ055, were synthesised in solution, characterised by 1H-, 13C- and 2D-NMR spectroscopy, and subsequently immobilised on the PEG-functionalised beads. Ligands SJ047 and SJ055 displayed apparent Kd values of 2.04x10-7 M and 1.91x10-8 M, respectively, against the ferritin. SJ047 and SJ055-functionalised resins were able to purify haemagglutinin (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-functionalised resins purified the recombinant antigens when spiked at known concentrations into HEK supernatants. All three strains, haemagglutinin (New Caledonia)-ferritin, haemagglutinin (California)-ferritin and haemagglutinin (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.
PMID: 28558204 DOI: 10.1021/acs.bioconjchem.7b00253