In situ detection of protein interactions for recombinant therapeutic enzymes

Mojtaba Samoudi, Chih Chung Kuo, Caressa M. Robinson, Km Shams-Ud-Doha, Song-Min Schinn, Stefan Kol, Linus Weiss, Sara Petersen Bjørn, Bjørn G. Voldborg, Alexandre Rosa Campos, Nathan E. Lewis*

*Corresponding author for this work

Research output: Contribution to journalJournal articleResearchpeer-review

Abstract

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.
Original languageEnglish
JournalBiotechnology and Bioengineering
Number of pages15
ISSN0006-3592
DOIs
Publication statusAccepted/In press - 2021

Keywords

  • BioID
  • Cell engineering
  • Disulfide bond
  • Secretory pathway
  • Therapeutic proteins

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