The use of regulatory mechanism of glycolytic enzvme gene expression for production of recombinant proteins in animal cells

氧气供应是培养动物细胞生产有用蛋白的主要问题之一，因此设计一种在低氧浓度下维持或提高人类治疗性重组蛋白高产率的系统是很重要的。在动物细胞中发现了许多缺氧诱导基因，大多数情况下的诱导是由于基因转录的缺氧激活。在这些基因中存在一个负责缺氧激活的公认序列（HRE=hypoxia-response enhancer），并且在动物细胞中广泛分布的一种蛋白质与这个响应缺氧的序列结合，可以激活启动子活性。乳酸脱氢酶A基因启动子在中国仓鼠卵巢（CHO）细胞中具有活性，而在缺氧条件下，邻近的HRE有效地刺激了启动子的活性。在此启动子/HRE的控制下，我们制备了一些产生重组人促红细胞生成素（Epo）的永久性CHO细胞系。当使用野生型HRE时，Epo的产生是高度缺氧诱导的，而当使用突变型HRE时，Epo的产生是不诱导的。在21%氧和2%氧条件下培养的细胞产生的Epo的体内外活性和糖基化程度差异不大。此外，强迫缺氧诱导因子-1α (HIF-1α)的表达在所有氧浓度下都能促进Epo的产生。这些结果表明，基于缺氧诱导基因转录的生物学策略提供了一种新的系统，即使在低氧浓度下也能保证高生产力。

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 under low oxygen concentrations.