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

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.