Quantitative analysis of transcription and translation in gene amplified Chinese hamster ovary cells on the basis of a kinetic model

The elevation of expression levels for secreted glycoproteins by gene ampli fication in mammalian cells shows a saturation behavior at high levels of g ene amplification. At high expression levels a drop in the secretion effici ency for the recombinant protein occurs (Schroder and Friedl, 1997), coinci ding with the appearance of misfolded protein in the cell. In this communic ation we investigated whether additional limitations exist at the levels of transcription and translation. Four Chinese hamster ovary (CHO) cell lines expressing different amounts of human antithrombin III (ATIII) were used a s a model system. A tenfold increase in the ATIII cDNA copy number from the lowest to the highest producing cell line coincided with a 38-fold increas e in ATIII mRNA levels, and an 80-fold increase in the amount of intracellu lar ATIII levels. The data was analyzed using a simple kinetic model. The f ollowing conclusions were derived: I. The transcriptional activity for the recombinant protein is not saturated. II. Translation itself is not saturat ed either, but may be downregulated as secretion efficiency drops. III. Two explanations for the previously reported drop in secretion efficiency for the recombinant protein with increasing expression level are possible: A. P rotein degradation is an alternative fate for translated ATIII and the frac tion of ATIII degraded after translation increases as expression level is i ncreased. B. Translation is downregulated as the secretory apparatus become s exhausted to maintain cell viability.