Establishment of DHFR-deficient HEK293 cells for high yield of therapeutic glycoproteins
Establishment of DHFR-deficient HEK293 cells for high yield of therapeutic glycoproteins
复制标题
建立 DHFR 缺陷型 HEK293 细胞以高产治疗性糖蛋白
DOI：
10.1016/j.jbiosc.2019.04.005
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发表时间：
2019-10-01
影响因子：
2.8
通讯作者：
Fujita, Morihisa
中科院分区：
文献类型：
--
作者：
Mensah, Emmanuel Osei;Guo, Xin-Yu;Fujita, Morihisa
由于蛋白质疗法可有效治疗人类疑难疾病，因此生物治疗剂的产量在过去三十年中急剧增加。中国仓鼠卵巢 (CHO) 细胞系是重组蛋白生产最常用的宿主细胞表达系统。使用二氢叶酸还原酶/甲氨蝶呤 (DHFR/MTX) 选择方法，从 DHFR 缺陷的 CHO 细胞中建立了用于重组蛋白生产的高产且稳定的克隆细胞系。人胚肾 293 (HEK293) 细胞是广泛用于蛋白质生产的替代宿主细胞。然而，大多数情况下，细胞用于瞬时表达，HEK293细胞中没有基因扩增系统。在本研究中，我们建立了 DHFR 缺陷的 HEK293 细胞系，以实现高产重组蛋白。我们使用 CRISPR/Cas9 系统在 MAN1A1/A2/B1/C1 四重敲除 HEK293 (QD-KO) 细胞中双重敲除 DHFR 和 DHFR2。 DHFR缺陷的QD-KO细胞通过DHFR/MTX基因扩增方法过表达溶酶体酸性脂肪酶和人免疫球蛋白G(1)恒定片段这两种蛋白。该方法通过增加 MTX 浓度，导致 DHFR 缺陷的 QD-KO 细胞中两种蛋白的表达显着增加。我们的系统可用于生产多种重组蛋白，包括治疗蛋白。 (C) 2019 年，日本生物技术协会。版权所有。
Since the use of protein therapeutics is effective for treating intractable human diseases, the production of biologic therapeutic agents has dramatically increased over the past three decades. The Chinese hamster ovary (CHO) cell lines are the most commonly used host cell expression system for recombinant protein production. High productive and stable clonal cell lines for recombinant protein production have been established from the DHFR-deficient CHO cell using the dihydrofolate reductase/methotrexate (DHFR/MTX) selection methods. Human embryonic kidney 293 (HEK293) cells are alternative host cells widely used for protein production. In most case, however, the cells are used for the transient expression, and there is no gene amplification system in HEK293 cells. In this study, we established a DHFR-deficient HEK293 cell line for the high yield of recombinant proteins. We doubly knocked out DHFR and DHFR2 in the MAN1A1/A2/B1/C1-quadruple knockout HEK293 (QD-KO) cells, using the CRISPR/Cas9 system. The DHFR-deficient QD-KO cells were used to overexpress two proteins, lysosomal acid lipase and the constant fragment of human immunoglobulin G(1) by the DHFR/MTX gene-amplification method. This method resulted in a dramatic increase in the two protein expressions in the DHFR-deficient QD-KO cells by increasing MTX concentration. Our system could be adopted in the production of several recombinant proteins including therapeutic proteins. (C) 2019, The Society for Biotechnology, Japan. All rights reserved.