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SHR Neuro Cancer Cardio Lipid Metab Microb

Zanker, AA; Stargardt, P; Kurzbach, SC; Turrina, C; Mairhofer, J; Schwaminger, SP; Berensmeier, S.
Direct capture and selective elution of a secreted polyglutamate-tagged nanobody using bare magnetic nanoparticles.
Biotechnol J. 2022; 17(5): e2100577 Doi: 10.1002/biot.202100577
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Co-authors Med Uni Graz: Schwaminger Sebastian
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Abstract:: 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.
Find related publications in this database (using NLM MeSH Indexing): Escherichia coli - genetics, metabolism; Magnetics - administration & dosage; Magnetite Nanoparticles - administration & dosage; Polyglutamic Acid - metabolism; Single-Domain Antibodies - administration & dosage
Find related publications in this database (Keywords): affinity peptide tag; downstream processing; magnetic iron oxide nanoparticles; PEG; secretion