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A novel, smaller scaffold for Affitins: Showcase with binders specific for EpCAM
Author(s) -
Kalichuk Valentina,
RenodonCornière Axelle,
Béhar Ghislaine,
Carrión Federico,
Obal Gonzalo,
Maillasson Mike,
Mouratou Barbara,
Préat Véronique,
Pecorari Frédéric
Publication year - 2018
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.26463
Subject(s) - thermostability , escherichia coli , recombinant dna , biochemistry , chemistry , affinities , protein engineering , enzyme , dna , extracellular , biology , microbiology and biotechnology , gene
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).