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Temperature Down‐Shift Modifies Expression of UPR‐/ERAD‐Related Genes and Enhances Production of a Chimeric Fusion Protein in CHO Cells
Author(s) -
Torres Mauro,
Akhtar Samia,
McKenzie Edward A.,
Dickson Alan J.
Publication year - 2021
Publication title -
biotechnology journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.144
H-Index - 84
eISSN - 1860-7314
pISSN - 1860-6768
DOI - 10.1002/biot.202000081
Subject(s) - endoplasmic reticulum associated protein degradation , unfolded protein response , chinese hamster ovary cell , fusion protein , endoplasmic reticulum , biology , microbiology and biotechnology , protein degradation , cell culture , gene expression , downregulation and upregulation , gene , biochemistry , recombinant dna , genetics
Low culture temperature enhances the cell‐specific productivity of Chinese hamster ovary (CHO) cells expressing varied recombinant (r‐) proteins, but the mechanisms remain unclear. Regulation of unfolded protein response (UPR) pathway genes, such as transcriptional regulatory factors and endoplasmic reticulum (ER)‐resident proteins, appear to be involved in the improvements of r‐protein production under low temperature conditions. The transcriptional regulation of UPR‐specific targets is studied in response to decreased culture temperature in relation to production of a difficult‐to‐express protein. A clonally‐derived CHO cell line expressing a chimeric fusion protein (human erythropoietin [hEPO] linked to a murine Fc region, hEPO‐Fc) is evaluated in terms of growth, metabolism, r‐protein production and UPR‐/ER associated degradation (ERAD)‐specific gene expression at standard (37 °C) and low (32 °C) temperature in batch and fed‐batch systems. Low temperature decreased peak cell density, improved viability, generated cell cycle arrest in the G1 phase and enhanced hEPO‐Fc expression in both batch and fed‐batch cultures. A low culture temperature significantly upregulated genes encoding UPR‐specific transcriptional activators ( xbp1s , ddit3 , and atf5 ) and ER‐resident proteins ( grp78 , grp94 , trib3 , and ero1α ), that are associated with folding and processing of proteins within the ER. Further, low culture temperature decreased expression of genes involved in ERAD ( edem3 , sels , herpud1 , and syvn1 ) indicating a decreased potential for protein degradation.