Intramolecular thioether cross-linking of therapeutic proteins to increase proteolytic stability
2014 (English)In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 15, no 14, 2132-2138 p.Article in journal (Other academic) Published
Protein-based pharmaceuticals typically display high selectivity and low toxicity, but are also characterized by low oral availability, mainly because of enzymatic degradation in the gastrointestinal tract and poor permeability across the intestinal wall. One way to increase the proteolytic stability of peptides and proteins is by intramolecular crosslinking, such as the introduction of disulfide bridges. However, disulfide bridges are at risk of thiol-disulfide exchange or reduction during production, purification, and/or therapeutic use, whereas thioether bridges are expected to be stable under the same conditions. In this study, thioether crosslinking was investigated for a 46 aa albumin-binding domain (ABD) derived from streptococcal protein G. ABD binds with high affinity to human serum albumin (HSA) and has been proposed as a fusion partner to increase the in vivo half-lives of therapeutic proteins. In the study, five ABD variants with single or double intramolecular thioether bridges were designed and synthesized. The binding affinity, secondary structure, and thermal stability of each protein was investigated by SPR-based biosensor analysis and CD spectroscopy. The proteolytic stability in the presence of the major intestinal proteases pepsin (found in the stomach) and trypsin in combination with chymotrypsin (found in pancreatin secreted to the duodenum by the pancreas) was also investigated. The most promising crosslinked variant, ABD_CL1, showed high thermal stability, retained high affinity in binding to HSA, and showed dramatically increased stability in the presence of pepsin and trypsin/chymotrypsin, compared to the ABD reference protein. This suggests that the intramolecular thioether crosslinking strategy can be used to increase the stability towards gastrointestinal enzymes.
Place, publisher, year, edition, pages
2014. Vol. 15, no 14, 2132-2138 p.
Albumin binding domain, solid phase peptide synthesis, proteolytic stability, oral delivery
Biochemistry and Molecular Biology
Research subject Biotechnology
IdentifiersURN: urn:nbn:se:kth:diva-141013DOI: 10.1002/cbic.201400002ISI: 000342807100017ScopusID: 2-s2.0-84908153975OAI: oai:DiVA.org:kth-141013DiVA: diva2:693810
FunderSwedish Research Council
Updated from "Manuscript" to "Article in Journal". QC 201411122014-02-052014-02-052014-11-12Bibliographically approved