Panoply™ Human THBD Over-expressing Stable Cell Line

Thrombomodulin (TM), encoded by the Thbd gene, plays key roles in processes such as coagulation, innate immunity, inflammation, and tumor cell proliferation. Here, researchers found that the presence of TM is highly correlated with recombinant protein yield in CHO cells producing Fc-fusion proteins. Knockout of Thbd resulted in an approximately 82% reduction in recombinant protein yield at the end of fed-batch culture, indicating that TM is essential for high-level production. Further investigation revealed that the decrease in yield was attributable to a significant reduction in recombinant protein mRNA levels. Re-expression of TM in the Thbd knockout cell line restored mRNA levels, confirming TM's role in maintaining transcription. Compared to untreated wild-type cells, knockout cells exhibited elevated phosphorylation levels of PKC, MEK, and ERK, alongside reduced phosphorylation of mTOR and AKT. Additionally, overexpression of Thbd led to a modest increase in c-Myc and Bcl2 expression, which appeared to mitigate the decline in cell viability during culture. Functional analysis of various TM domains demonstrated that the N-terminal lectin-like domain and the C-terminal cytoplasmic tail had a more significant impact on recombinant protein production than other regions.

In this study, researchers successfully generated stable CHO-Fc-WT cell lines with either Thbd gene overexpression (OE) or knockout (KO). The expression of thrombomodulin (TM)-encoded by the Thbd gene-was confirmed via immunofluorescence (Figure 1A) and Western blot (Figure 1B). Western blot analysis (Figure 1B) revealed that TM expression levels in Thbd-overexpressing cells (OE cells) were approximately 13.3 times higher than in WT cells, whereas levels in the KO cell line dropped to below 10% of those in WT cells (Figure 1C); these results were consistent with the immunofluorescence findings. During fed-batch culture (Figure 1D), the viable cell density of the OE cell line remained lower than that of the WT and KO lines until day 9, suggesting that TM overexpression might inhibit CHO cell growth during the early stages of culture. The KO cell line exhibited the highest growth rate throughout the culture period, indicating that the absence of TM promoted CHO cell proliferation. Conversely, the OE cell line maintained higher cell viability compared to the WT line (Figure 1D and E); consequently, its viable cell density surpassed that of the WT line in the later stages of culture (Figure 1F), thereby supporting sustained recombinant protein secretion.

Figure 1. Evaluation of monoclonal TM knockout and overexpression CHO-Fc-WT cell lines. (Ma X, et al., 2025)

Compared to WT cells, recombinant protein yields from the KO cell line decreased by 84.1% and 82.04% on days 9 and 11, respectively (Figure 1G). Although the KO cell line maintained higher viable cell counts than the WT and OE lines throughout the process (Figure 1D and F)-a factor typically expected to yield higher protein output-these results strongly suggest that the presence of TM is crucial for efficient recombinant protein production in CHO cells. However, unlike results observed in previous transient treatment experiments, stable TM overexpression in the OE cell line did not result in a higher final recombinant protein yield (Figure 1G, day 11); instead, the yield showed only a marginal increase of 10.4%. This indicates that a significant increase in TM molecule abundance on the cell membrane does not necessarily enhance recombinant protein production.