Design of a Cost-Effective Strategy for Secretory Production of Recombinant CCN Proteins By Mammalian Cells

CCN proteins (CCNs), a family of secreted extracellular matrix (ECM)-associated proteins, constitutes six members (CCN1-CCN6). CCN protein family plays the important role in various pathophysiological conditions such as human cancers and cardiovascular diseases. In human tumorigenesis, clinical expression levels of CCNs have been shown to be correlated with malignancies of several human cancer types. In cellular levels, CCNs can modulate multiple cellular activities, including cancer cell migration, invasion, stem-like properties that drive cancer initiation, and metastasis processes.  In addition, studies indicate that members of CCNs, particularly the CCN1 and CCN5, can act as the conditional tumor suppressors in certain cancers. This suggests that recombinant CCNs-based protein products may have potential of being developed as therapeutic agents. Our aim is to utilize mammalian cell expression system to establish a cost-effective protocol for production of recombinant CCN proteins, followed by testing the anti-cancer effects of the CCNs-derived proteins.

 In the present study, we utilized the lentivirus-based transgene vector to express CCN1 and CCN5 proteins by human HEK293 cells. Following viral infection and antibiotic selection, the stable CCNs-expressing cells were further enriched by cell line selection and isolation. High-level expression and secretion of recombinant proteins were optimized by the processes relevant to metabolic engineering of mammalian host cells, including addition of sodium butyrate, change of medium osmolarity, as well as hypoxia-reoxygenation cultivation procedure. Finally, the recombinant CCN proteins were successfully collected from serum-free media, and then purified by affinity chromatography. The recombinant CCN proteins were proved to be bioactive by the effective intervention in inhibiting cancer cell malignancy such as reduction of cellular mobility and impaired cancer stemness.

 Together, we have successfully established a mammalian cell expression system to produce CCNs-derived protein products of therapeutic potential. The CCNs-related proteins may be applied to target relevant human diseases in the near future.