A new biological strategy for high productivity of recombinant proteins in animal cells by the use of hypoxia-response enhancer

Abstract

Oxygen supply is one of the major problems in the production of useful proteins by cultured animal cells and therefore it is of importance to devise a system by which a high productivity of human therapeutic recombinant proteins can be maintained or enhanced under low oxygen concentrations. A number of hypoxia‐inducible genes have been found in animal cells and the induction in most cases is due to hypoxic activation of the gene transcription. A consensus sequence (HRE = hypoxia‐response enhancer) responsible for the hypoxic activation exists in these genes and the binding of a protein, which is widely distributed in animal cells, to this sequence responding to hypoxia activates the promoter activity. The promoter of lactate dehydrogenase A gene is active in Chinese hamster ovary (CHO) cells and the vicinal HRE stimulates the promoter activity efficiently in hypoxia. We have prepared a number of permanent CHO cell lines producing recombinant human erythropoietin (Epo) under control of this promoter/HRE. Epo production was highly hypoxia‐inducible when the wild‐type of HRE was used but uninducible when the mutant HRE was used. There was little difference in the in vitro and in vivo activities, and glycosylation between Epo produced by the cells cultured in 21% and 2% oxygen. Furthermore, forced expression of hypoxia‐inducible factor‐1α (HIF‐1α) enhanced Epo production in all oxygen concentrations. These results indicate that a biological strategy based on the hypoxic induction of gene transcription provides a novel system which guarantees a high productivity even uner low oxygen concentrations. © 2000 John Wiley & Sons, Inc. Biotechnol Bioeng 67: 157–164, 2000.

Improved production of recombinant human antithrombin III in Chinese hamster ovary cells by ATF4 overexpression

“…To attain high productivity of a recombinant protein in mammalian cells, attention has been paid to development of cell lines with a high specific rate of protein production (Birch and Racher, 2006). Among techniques used for the cell line development were enhanced gene expression under strong promoters (Schwartz et al, 1991), transcriptional activation using enhancer sequences (Masuda et al, 2000), and gene amplification and cell line screening (Kellems, 1991). These techniques have made a significant contribution to the cell line improvement at the transcriptional level.…”

Enhancing recombinant protein production in human cell lines with a constitutive transport element and mRNA export proteins

“…EPO activity in the culture supernatant was measured by a sandwich enzymelinked immunosorbent assay as described previously (Masuda et al, 1999(Masuda et al, , 2000a. ␤-Galactosidase activity was measured as described previously (Masuda et al, 2000b). The total protein content was measured using a protein assay kit (Nacalai, Kyoto, Japan).…”

Transcriptional Targeting for Ovarian Cancer Gene Therapy

“…The matrix metalloprotease pump 1 (MMP7) is also frequently overexpressed in ovarian tumors [69]. Other genes, involved in tumor invasion and angiogenesis are being increas- [76] ingly elucidated in the context of ovarian cancer. For example, the urokinase-type plasminogen activator (uPA) and its receptor (uPAR), implicated in the cleavage of extracellular matrix proteins, are elevated in ovarian carcinoma cells [70].…”

“…An alternative transcriptional strategy to target refractory tumor cells capitalizes on the use of promoters that are induced under certain conditions present in the tumor environment, (such as hypoxia) that hinder the efficacy of chemotherapy and radiotherapy. One such TSP is the promoter of the lactate dehydrogenase gene which is efficiently induced by the associated hypoxia-response enhancer (HRE) in ovarian cancer cell lines [76].…”