Thrombomodulin is essential for recombinant Fc-fusion protein production in Chinese hamster ovary cells via multiple signaling pathways.

BACKGROUND: Chinese hamster ovary (CHO) cells are the predominant cell line used for biotherapeutic production. To reduce the cost of therapeutic recombinant proteins produced in CHO cells, efforts have been made over decades to improve overall yield through media and process optimization, as well as genetic engineering aimed at enhancing cell proliferation or productivity. Within an intricate cellular framework, such as the CHO cell system, it is indisputable that numerous genes with seemingly disparate functions may be involved in the process of expressing recombinant proteins. RESULTS: Thrombomodulin (TM), encoded by the Thbd gene, is primarily known for its roles in coagulation, innate immunity, inflammation, and tumor cell proliferation. Our research was the first to reveal that the presence of thrombomodulin is highly correlated with recombinant protein production in CHO cells producing an Fc-fusion protein. Knocking out Thbd resulted in approximately an 82% reduction in recombinant protein yield by the end of fed-batch culture, indicating that TM is essential for efficient production. Further investigation revealed that this loss was due to a dramatic reduction in mRNA levels of the recombinant protein. Re-expression of TM in the Thbd-knockout cell line restored mRNA levels, confirming TM's role in maintaining transcription. Phosphorylation levels of PKC, MEK, and ERK were elevated in the knockout cells compared to untreated wild-type cells, whereas phosphorylation of mTOR and AKT was decreased. Additionally, overexpression of Thbd led to moderate increases in c-Myc and Bcl2 expression, which appeared to slow the decline in cell viability during cultivation. Functional analyses of different TM domains revealed that both the N-terminal lectin-like domain and the C-terminal cytoplasmic tail have greater impacts on recombinant protein production than the other regions. CONCLUSIONS: This study demonstrates the essential role of thrombomodulin in recombinant Fc-fusion protein production in Chinese hamster ovary cells, reveals novel biological functions of thrombomodulin, and expands our understanding of the complex cellular machinery underlying recombinant protein expression in CHO cells.