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Intramolecular Thioether Crosslinking to Increase the Proteolytic Stability of Affibody Molecules
Author(s) -
Nilsson Anders,
Lindgren Joel,
Eriksson Karlström Amelie
Publication year - 2017
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.201700350
Subject(s) - thioether , intramolecular force , chemistry , proteolytic enzymes , molecule , combinatorial chemistry , biophysics , polymer chemistry , stereochemistry , organic chemistry , biology , enzyme
Protein therapeutics suffer from low oral bioavailability, mainly due to poor membrane permeability and digestion by gastrointestinal proteases. To improve proteolytic stability, intramolecular thioether crosslinks were introduced into a three‐helix affibody molecule binding the human epidermal growth factor receptor (EGFR). Solid‐phase peptide synthesis was used to produce an unmodified control protein domain and three different crosslinked protein domain variants: one with a thioether crosslink between the N‐terminal lysine residue and a cysteine residue in the second loop region (denoted K4), a second with a crosslink between the C‐terminal lysine residue and a cysteine residue in the first loop region (denoted K58), and a third with crosslinks in both positions (denoted K4K58). Circular dichroism (CD) and surface‐plasmon‐resonance‐based (SPR‐based) biosensor studies of the protein domains showed that the three‐helix structure and high‐affinity binding to EGFR were preserved in the crosslinked protein domains. In vitro digestion by gastrointestinal proteases demonstrated that the crosslinked protein domains showed increased stability towards pepsin and towards a combination of trypsin and chymotrypsin.