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In situ detection of protein interactions for recombinant therapeutic enzymes
Author(s) -
Samoudi Mojtaba,
Kuo ChihChung,
Robinson Caressa M.,
ShamsUdDoha Km,
Schinn SongMin,
Kol Stefan,
Weiss Linus,
Petersen Bjorn Sara,
Voldborg Bjorn G.,
Rosa Campos Alexandre,
Lewis Nathan E.
Publication year - 2021
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.27621
Subject(s) - recombinant dna , protein disulfide isomerase , protein folding , secretion , biochemistry , secretory pathway , secretory protein , chemistry , folding (dsp implementation) , glycosylation , disulfide bond , biology , microbiology and biotechnology , gene , cell , electrical engineering , golgi apparatus , engineering
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 posttranslational 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.