TY - JOUR
T1 - Compartmentalized Proteomic Profiling Outlines the Crucial Role of the Classical Secretory Pathway during Recombinant Protein Production in Chinese Hamster Ovary Cells
AU - Pérez-Rodriguez, Saumel
AU - Wulff, Tune
AU - Voldborg, Bjørn G.
AU - ALTAMIRANO GOMEZ, CLAUDIA VICTORIA
AU - Trujillo-Roldán, Mauricio A.
AU - Valdez-Cruz, Norma A.
N1 - Funding Information:
Saumel Pérez-Rodriguez is a doctoral student from Programa de Doctorado en Ciencias Biomédicas, Universidad Nacional Autónoma de México (UNAM) and received fellowship number 396822 from “Consejo Nacional de Ciencia y Tecnología” CONACYT. We thank Dr. Octavio T Ramírez for the donation of one of the cell lines used in this work and the critical review of the manuscript. We thank Dr. Paola Toledo-Ibelles and Inolab Especialistas en Servicio S.A. de C.V. for their support in cell culture, to BSc. Sandra Guerrero-Peralta for her contribution to cell culture and antibody purification, and to Dr. Marcela Lizano Soberón and Dr. Alejandro Alagón Cano for their advice and critical review of methodology and experimental results. We also want to extend our gratitude to DNASU repository for providing hIL8 coding plasmid and to Sartorius De México and S.A. De C.V. for their technical assistance, as well as to Nova Biomedicals for providing access to BioProfile FLEX2 Automated Cell Culture Analyzer. This work was supported by “Programa de Apoyo a Proyectos de Investigación e Innovación Tecnológica, Universidad Nacional Autónoma de México” (PAPIIT-UNAM IN210419, IT-200719, IN-208415). This work was partially funded by the National Council for Science and Technology (CONACYT, Consejo Nacional de Ciencia y Tecnología, 247473, 220795). The work carried out at Novo Nordisk Foundation Center for Biosustainability was founded by Novo Nordisk foundation (NNF10CC1016517). Funding sources had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Publisher Copyright:
© 2021 The Authors. Published by American Chemical Society.
PY - 2021
Y1 - 2021
N2 - Different cellular processes that contribute to protein production in Chinese hamster ovary (CHO) cells have been previously investigated by proteomics. However, although the classical secretory pathway (CSP) has been well documented as a bottleneck during recombinant protein (RP) production, it has not been well represented in previous proteomic studies. Hence, the significance of this pathway for production of RP was assessed by identifying its own proteins that were associated to changes in RP production, through subcellular fractionation coupled to shot-gun proteomics. Two CHO cell lines producing a monoclonal antibody with different specific productivities were used as cellular models, from which 4952 protein groups were identified, which represent a coverage of 59% of the Chinese hamster proteome. Data are available via ProteomeXchange with identifier PXD021014. By using SAM and ROTS algorithms, 493 proteins were classified as differentially expressed, of which about 80% was proposed as novel targets and one-third were assigned to the CSP. Endoplasmic reticulum (ER) stress, unfolded protein response, calcium homeostasis, vesicle traffic, glycosylation, autophagy, proteasomal activity, protein synthesis and translocation into ER lumen, and secretion of extracellular matrix components were some of the affected processes that occurred in the secretory pathway. Processes from other cellular compartments, such as DNA replication, transcription, cytoskeleton organization, signaling, and metabolism, were also modified. This study gives new insights into the molecular traits of higher producer cells and provides novel targets for development of new sub-lines with improved phenotypes for RP production.
AB - Different cellular processes that contribute to protein production in Chinese hamster ovary (CHO) cells have been previously investigated by proteomics. However, although the classical secretory pathway (CSP) has been well documented as a bottleneck during recombinant protein (RP) production, it has not been well represented in previous proteomic studies. Hence, the significance of this pathway for production of RP was assessed by identifying its own proteins that were associated to changes in RP production, through subcellular fractionation coupled to shot-gun proteomics. Two CHO cell lines producing a monoclonal antibody with different specific productivities were used as cellular models, from which 4952 protein groups were identified, which represent a coverage of 59% of the Chinese hamster proteome. Data are available via ProteomeXchange with identifier PXD021014. By using SAM and ROTS algorithms, 493 proteins were classified as differentially expressed, of which about 80% was proposed as novel targets and one-third were assigned to the CSP. Endoplasmic reticulum (ER) stress, unfolded protein response, calcium homeostasis, vesicle traffic, glycosylation, autophagy, proteasomal activity, protein synthesis and translocation into ER lumen, and secretion of extracellular matrix components were some of the affected processes that occurred in the secretory pathway. Processes from other cellular compartments, such as DNA replication, transcription, cytoskeleton organization, signaling, and metabolism, were also modified. This study gives new insights into the molecular traits of higher producer cells and provides novel targets for development of new sub-lines with improved phenotypes for RP production.
UR - http://www.scopus.com/inward/record.url?scp=85106639095&partnerID=8YFLogxK
U2 - 10.1021/acsomega.0c06030
DO - 10.1021/acsomega.0c06030
M3 - Article
AN - SCOPUS:85106639095
JO - ACS Omega
JF - ACS Omega
SN - 2470-1343
ER -