Reshaping nanobodies for affinity purification on protein a

Maxine Crauwels, Nele Van Vaerenbergh, Neeme Benedict Kulaya, Cécile Vincke, Matthias D'Huyvetter, Nick Devoogdt, Serge Muyldermans, Catarina Xavier

Onderzoeksoutput: Articlepeer review

6 Citaten (Scopus)

Samenvatting

Nanobodies (Nbs) are 15 kDa recombinant, single-domain, antigen-specific fragments derived from heavy-chain only antibodies (HCAbs) occurring naturally in species of Camelidae. The beneficial properties of Nbs make them suitable tracers for diagnostic and therapeutic purposes. Whereas Nbs with a terminal hexa-histidine tag (His-tag) are easily purified via immobilized metal affinity chromatography, previous studies revealed a negative impact of the His-tag on the biodistribution of Nb-based tracers. Thus, it is important to develop alternative purification methods for Nbs without a His-tag. Protein A (SpA), a surface protein of Staphylococcus aureus, binds the Fc-region of IgG molecules and also to a lesser extent human heavy chain family-3 variable (VH) regions. Nbs also belong to this VH family, although many fail to be recognized by SpA. Here it is demonstrated that non-SpA binding Nbs can be mutagenized for purification by SpA affinity chromatography and that these Nb variants retain their thermostability and antigen affinity, while biodistribution remains unaffected.

Originele taal-2English
Pagina's (van-tot)20-28
Aantal pagina's9
TijdschriftNew Biotechnology
Volume57
DOI's
StatusPublished - 25 jul 2020

Bibliografische nota

Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.

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