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A novel, smaller scaffold for Affitins: Showcase with binders specific for EpCAM.

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Kalichuk, Valentina [UCL] Renodon-Cornière, Axelle Béhar, Ghislaine Carrión, Federico Obal, Gonzalo Préat, Véronique [UCL] Pecorari, Frédéric

Affitins are highly stable engineered affinity proteins, originally derived from Sac7d and Sso7d, two 7 kDa DNA-binding polypeptides from Sulfolobus genera. Their efficiency as reagents for intracellular targeting, enzyme inhibition, affinity purification, immunolocalization, and various other applications has been demonstrated. Recently, we have characterized the 7 kDa DNA-binding family, and Aho7c originating from Acidianus hospitalis was shown to be its smallest member with thermostability comparable to those of Sac7d and Sso7d. Here, after four rounds of selection by ribosome display against the human recombinant Epithelial Cell Adhesion Molecule (hrEpCAM), we obtained novel Aho7c-based Affitins. The binders were expressed in soluble form in Escherichia coli, displayed high stability (up to 74°C; pH 0-12) and were shown to be specific for the hrEpCAM extracellular domain with picomolar affinities (KD  = 110 pM). Thus, we propose Aho7c as a good candidate for the creation of Affitins with a 10% smaller size than the Sac7d-based ones (60 vs. 66 amino acids).

Document type Article de périodique (Journal article) – Article de recherche
Publication date 2018
Language Anglais
Journal information "Biotechnology and Bioengineering" - Vol. 115, p. 290-299 (2018)
Peer reviewed yes
Publisher JohnWiley & Sons, Inc. (Hoboken)
issn 0006-3592
e-issn 1097-0290
Publication status Soumis
Affiliation UCL - SSS/LDRI - Louvain Drug Research Institute
Keywords Affitin ; Aho7c ; EpCAM protein engineering ; Sac7d ; ribosome display
Links
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Bibliographic reference Kalichuk, Valentina ; Renodon-Cornière, Axelle ; Béhar, Ghislaine ; Carrión, Federico ; Obal, Gonzalo ; et. al. A novel, smaller scaffold for Affitins: Showcase with binders specific for EpCAM.. In: Biotechnology and Bioengineering, Vol. 115, p. 290-299 (2018)
Permanent URL http://hdl.handle.net/2078.1/192276