Efficiency of translation and post-translation regulatory genes in optimization of tissue plasminogen activator gene expression

Chinese hamster ovary (CHO) cells are the most commonly used host system for the expression of high quality recombinant proteins. Different strategies such as generation of more efficient expression vectors and establishment of genetically engineered host cells have been employed to improve recombinant protein expression in this system. Here the cell line engineering strategy based on the phosphorylation resistant variants of eukaryotic initiation factor 2 alpha(eIF2α) and ceramide transfer protein(CERT), key mediators of protein translation and secretion, have been used to develop improved CHO host cells. The CERT S132A, peIF2α S51A and tissue plasminogen activator (t-PA) were cloned in suitable expression vectors. CERT S132A and peIF2α S51A stable cell pools were generated and the efficacy of the cells for transient expression of t-PA was evaluated in comparison to un-modified CHO cells. Integration of CERT S132A and peIF2α S51A expression vectors in CHO cell genome confirmed by PCR analysis of genomic DNA from stable cells. Moreover, the specific bands for CERT S132A and peIF2α S51A proteins were observed in Western blot analysis. Expression of CERT S132A gene increased expression of t-PA by 50%. However no positive effect on t-PA expression was observed in the peIF2α S51A stable cell pools. This study showed the efficiency of CERT S132Abased cell line engineering approach for the development of optimized CHO host cells.