A novel, smaller scaffold for Affitins: Showcase with binders specific for EpCAM. Issue 2 (6th November 2017)

• Record Type: Journal Article
• Authors: Kalichuk, Valentina; Renodon‐Cornière, Axelle; Béhar, Ghislaine; Carrión, Federico; Obal, Gonzalo; Maillasson, Mike; Mouratou, Barbara; Préat, Véronique; Pecorari, Frédéric
• Is Part Of: Biotechnology and bioengineering. Volume 115:Issue 2(2018)
• Page Start: 290
• Page End: 299
• Publication Date: 2017-11-06
• Subjects: Affitin -- Aho7c -- EpCAM protein engineering -- Sac7d -- ribosome display; Biotechnology -- Periodicals; Bioengineering -- Periodicals; 660.6
• DOI: 10.1002/bit.26463
• Languages: English
• ISSNs: 0006-3592

Abstract

Abstract: 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 ( K D = 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). Abstract : Affitins are highly stable engineered affinity proteins, originally derived from Sac7d and Sso7d polypeptides belonging to the archaeal Sul7d family. Recently, we have characterized this family and identified Aho7c as its smallest member. Here, Aho7c‐based Affitins specific for EpCAM were obtained after selection by ribosome display. They showed high stability (up to 74°C; pH 0–12) and affinities ( K D = 110 pM), and thus Aho7c is proposed as a good candidate for the creation of Affitins with a 10% smaller size than the Sac7d‐based ones.