Increase in cysteine-mediated multimerization under attractive protein-protein interactions

The CASPON enzyme became an interesting enzyme for fusion protein processing because it generates an authentic N-terminus. However, the high cysteine content of the CASPON enzyme may induce aggregation via disulfide-bond formation, which can reduce enzymatic activity and be considered a critical qua...

Full description

Saved in:
Bibliographic Details
Published in:Preparative biochemistry & biotechnology 2023-09, Vol.53 (8), p.891-905
Main Authors: Jakob, Leo A., Mesurado, Tomás, Jungbauer, Alois, Lingg, Nico
Format: Article
Language:English
Subjects:
aggregation
Ammonium
Ammonium sulfate
Bonding
Caspase
Catalysis
Chromatography
Cysteine
disulfide
Dithiothreitol
downstream processing
Enzymatic activity
Enzymes
Fusion protein
multimerization
N-Terminus
Proteases
Protein interaction
Protein purification
protein stability
protein unfolding
protein-protein interaction
Proteins
Quality management
Size exclusion chromatography
Solutes
Stationary phase
Citations: Items that this one cites
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:The CASPON enzyme became an interesting enzyme for fusion protein processing because it generates an authentic N-terminus. However, the high cysteine content of the CASPON enzyme may induce aggregation via disulfide-bond formation, which can reduce enzymatic activity and be considered a critical quality attribute. Different multimerization states of the CASPON enzyme were isolated by preparative size exclusion chromatography and analyzed with respect to multimerization propensity and enzymatic activity. The impact of co-solutes on multimerization was studied in solution and in adsorbed state. Furthermore, protein-protein interactions in the presence of different co-solutes were measured by self-interaction chromatography and were then correlated to the multimerization propensity. The dimer was the most stable and active species with 50% higher enzymatic activity than the tetramer. Multimerization was mainly governed by a cysteine-mediated pathway, as indicated by DTT-induced reduction of most caspase multimers. In the presence of ammonium sulfate, attractive protein-protein interactions were consistent with those observed for higher multimerization when the cysteine-mediated pathway was followed. Multimerization was also observed under attractive conditions on a chromatographic stationary phase. These findings corroborate common rules to perform protein purification with low residence time to avoid disulfide bond formation and conformational change of the protein upon adsorption.
ISSN:1082-6068
1532-2297
DOI:10.1080/10826068.2022.2158471