Journal article
In situ detection of protein interactions for recombinant therapeutic enzymes
University of California at San Diego1
Sanford Burnham Prebys Medical Discovery Institute2
Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark3
University of Tübingen4
Department of Biotechnology and Biomedicine, Technical University of Denmark5
CHO Core, Translational Management, Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark6
Despite their therapeutic potential, many protein drugs remain inaccessible to patients since they are difficult to secrete. Each recombinant protein has unique physicochemical properties and requires different machinery for proper folding, assembly, and post-translational modifications (PTMs). Here we aimed to identify the machinery supporting recombinant protein secretion by measuring the protein-protein interaction (PPI) networks of four different recombinant proteins (SERPINA1, SERPINC1, SERPING1 and SeAP) with various PTMs and structural motifs using the proximity-dependent biotin identification (BioID) method.
We identified PPIs associated with specific features of the secreted proteins using a Bayesian statistical model, and found proteins involved in protein folding, disulfide bond formation and N-glycosylation were positively correlated with the corresponding features of the four model proteins. Among others, oxidative folding enzymes showed the strongest association with disulfide bond formation, supporting their critical roles in proper folding and maintaining the ER stability.
Knockdown of disulfide-isomerase PDIA4, a measured interactor with significance for SERPINC1 but not SERPINA1, led to the decreased secretion of SERPINC1, which relies on its extensive disulfide bonds, compared to SERPINA1, which has no disulfide bonds. Proximity-dependent labeling successfully identified the transient interactions supporting synthesis of secreted recombinant proteins and refined our understanding of key molecular mechanisms of the secretory pathway during recombinant protein production.
This article is protected by copyright. All rights reserved.
Language: | English |
---|---|
Year: | 2021 |
Pages: | 890-904 |
ISSN: | 10970290 and 00063592 |
Types: | Journal article |
DOI: | 10.1002/bit.27621 |
ORCIDs: | Kol, Stefan , Petersen Bjørn, Sara , Voldborg, Bjørn G. and 0000-0001-7700-3654 |