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

Research output: Contribution to journalArticlepeer-review

3 Citations (Scopus)


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.

Original languageEnglish
Pages (from-to)20-28
Number of pages9
JournalNew Biotechnology
Publication statusPublished - 25 Jul 2020

Bibliographical note

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


  • Affinity chromatography
  • Nanobody
  • Protein A.


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