Direct capture and selective elution of a secreted polyglutamate‐tagged nanobody using bare magnetic nanoparticles

• Published in: Biotechnology journal 2022-05, Vol.17 (5), p.e2100577-n/a
• Main Authors: Zanker, Alexander A., Stargardt, Patrick, Kurzbach, Sophie C., Turrina, Chiara, Mairhofer, Juergen, Schwaminger, Sebastian P., Berensmeier, Sonja
• Format: Article
• Language: English
• Subjects: affinity peptide tag; downstream processing; magnetic iron oxide nanoparticles; PEG; secretion
• ISSN: 1860-6768; 1860-7314
• DOI: 10.1002/biot.202100577

Background The secretion and direct capture of proteins from the extracellular medium is a promising approach for purification, thus enabling integrated bioprocesses. Major Results We demonstrate the secretion of a nanobody (VHH) to the extracellular medium (EM) and its direct capture by bare, non‐functionalized magnetic nanoparticles (MNPs). An ompA signal peptide for periplasmic localization, a polyglutamate‐tag (E8) for selective MNP binding, and a factor Xa protease cleavage site were fused N‐terminally to the nanobody. The extracellular production of the E8‐VHH (36 mg L–1) was enabled using a growth‐decoupled Escherichia coli‐based expression system. The direct binding of E8‐VHH to the bare magnetic nanoparticles was possible and could be drastically improved up to a yield of 88% by adding polyethylene glycol (PEG). The selectivity of the polyglutamate‐tag enabled a selective elution of the E8‐VHH from the bare MNPs while raising the concentration factor (5x) and purification factor (4x) significantly. Conclusion Our studies clearly show that the unique combination of a growth‐decoupled E. coli secretion system, the polyglutamate affinity tag, non‐functionalized magnetic nanoparticles, and affinity magnetic precipitation is an innovative and novel way to capture and concentrate nanobodies. Graphical and Lay Summary A polyglutamate‐tagged nanobody (E8‐VHH) was extracellularly expressed in a modified E. coli strain. By adding non‐functionalized magnetic nanoparticles (MNP), the E8‐VHH was captured and selectively eluted, while the yield was improved by the addition of PEG and pH adjustment.