Construction of engineered CHO strains for high-level production of recombinant proteins
Construction of engineered CHO strains for high-level production of recombinant proteins
复制标题
DOI：
10.1007/s00253-002-1134-1
复制
发表时间：
2002-12-01
影响因子：
5
通讯作者：
Shibui, T
中科院分区：
文献类型：
--
作者：
Kito, M;Itami, S;Shibui, T
我们构建了工程CHO菌株，可用于通过基因靶向高水平生产外源蛋白。在用携带loxP-绿色荧光蛋白（GFP）融合基因和DHFR基因的质粒转染DHFR缺陷型CHO细胞后，我们通过荧光强度筛选菌落。我们选择了16个克隆，表达高水平的GFP和携带一个拷贝的质粒在他们的染色体和治疗他们与氨甲喋呤（MTX），以检查他们的能力DHFR介导的基因扩增。两个克隆MK 1和MK2在基因扩增后显示GFP表达增加。在这些克隆中，loxP-GFP基因整合在染色体中转录活性的、DHFR介导的、基因可扩增的基因座处。通过Cre重组酶介导的位点特异性重组，构建了携带loxP融合潮霉素抗性基因的基因靶向载体，以靶向染色体loxP中的所需基因。利用这种细胞载体系统，我们可以重复地获得高产量的重组蛋白的基因打靶和基因扩增。在人单克隆抗体生产中，在MK2中的loxP的基因靶向和用MTX的基因扩增后，MTX抗性菌落显示出高水平的抗体生产。最具生产力的克隆能够产生160毫克/升，在7天内在低蛋白培养基中的转瓶。
We constructed engineered CHO strains that can be used for high-level production of foreign proteins by gene-targeting. After transfecting dihydroforate reductase (DHFR)-deficient CHO cells with a plasmid carrying a loxP-green fluorescent protein (GFP) fusion gene and a DHFR gene, we screened colonies by fluorescent intensity. We selected 16 clones that expressed high levels of GFP and carried one copy of the plasmid in their chromosomes and treated them with methotrexate (MTX) to examine their ability for DHFR-mediated gene amplification. Two clones, MK1 and MK2, showed increased GFP expression upon gene amplification. In those clones, the loxP-GFP gene was integrated at a transcription-active, DHFR-mediated, gene-amplifiable locus in the chromosomes. A gene-targeting vector, carrying a loxP-fused hygromycin-resistance gene, was constructed to target desired genes in chromosomal loxP by Cre recombinase-mediated site-specific recombination. Using this cell-vector system, we could reproducibly obtain high producers of recombinant proteins by gene-targeting and gene amplification. In human monoclonal antibody production, after gene-targeting of loxP in MK2 and gene amplification with MTX, the MTX-resistant colonies showed high levels of antibody production. The most productive clone was able to produce 160 mg/l in 7 days in a low-protein medium in a spinner-flask.