Enhancement of cellular productivity for a recombinant protein by a combination of promoter activation and gene amplification

Abstract

We have developed an oncogene activated production (OAP) system using amplified ras oncogene to activate the CMV promoter controlling the foreign gene in mammalian cells (Yano, T. et al., 1994). CHO cells were demonstrated to be suited for the OAP system (Perry, S. et al., 1996). Here we show that very high level production of recombinant protein can be achieved by combining the OAP system with the glutamine synthetase (GS) gene amplification system. The human interleukin 6 (hIL-6) gene regulated by the human CMV promoter was inserted into a GS-mini-gene expression plasmid pEE14. A highly productive host CHO cell line, ras-clone I containing amplified ras oncogene was further transfected with the plasmid expressing both hIL-6 and GS mini-gene, and selected with methionine sulphoximine (MSX) in G-DME medium supplemented with 10% dialyzed fetal bovine serum. We could establish a hEL-6 hyper-producing cell line DRGI-29 derived from ras-clone I, which exhibited a peak productivity value of about 40 ¼:g hIL-6/106 cells/day through a combination of the OAP system and GS gene amplification system. The cellular productivity of DRGI-29 cells was about 13 times higher than the control hIL-6-producing cells derived from normal CHO cells whose hIL-6 gene was amplified by GS gene amplification system, and about 5 times higher than I13 cells established by the OAP system, which contains amplified ras oncogene and non-amplified hIL-6 gene (Perry, S. et al., 1996). When DRGI-29 cells were cultivated for 30 days, the accumulation rate of about 80 μg hIL-6/ml/3days on 9th day. Whereas, the maximum productivity was achieved after long-term culture in the case of I13 cells (Perry, S. et al., 1996). These results indicate that this OAP and GS hybrid system enables the efficient and rapid establishment of recombinant protein hyper-producing cell lines.