Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu 214122, China.
Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu 214122, China.
J Biosci Bioeng. 2021 Mar;131(3):225-233. doi: 10.1016/j.jbiosc.2020.10.004. Epub 2020 Nov 4.
Most biopharmaceutical proteins are produced in mammalian cells because they have the advantageous capacity for protein folding, assembly, and posttranslational modifications. To satisfy the increasing demand for these proteins for clinical purposes and studies, traditional methods to improve protein productivity have included gene amplification, host cell engineering, medium optimization, and screening methods. However, screening and selection of high-producing cell lines remain complex and time consuming. In this study, we established a glycosylphosphatidylinositol (GPI)-anchored protein with a selenocysteine (GPS) system to select cells producing high levels of target secretory proteins. Recombinant lysosomal acid lipase (LIPA) and α-galactosidase A (GALA) were fused with a GPI attachment signal sequence and a selenocysteine insertion sequence after an in-frame UGA codon. Under these conditions, most of the recombinant proteins were secreted into the culture medium, but some were found to be GPI-anchored proteins on the cell surface. When sodium selenite was supplied into the culture medium, the amount of GPI-anchored LIPA and GALA was increased. High-expressing cells were selected by detecting surface GPI-anchored LIPA. The GPI-anchored protein was then eliminated by knocking out the GPI biosynthesis gene PIGK, in these cells, all LIPA was in secreted form. Our system provides a promising method of isolating cells that highly express recombinant proteins from large cell populations.
大多数生物制药蛋白都是在哺乳动物细胞中生产的，因为它们具有有利的蛋白质折叠、组装和翻译后修饰能力。为了满足临床用途和研究对这些蛋白质日益增长的需求，传统的提高蛋白质产量的方法包括基因扩增、宿主细胞工程、培养基优化和筛选方法。然而，高产细胞系的筛选和选择仍然复杂且耗时。在本研究中，我们建立了一种带有糖基磷脂酰肌醇（GPI）锚定和硒代半胱氨酸（GPS）系统的糖基化磷脂酰肌醇（GPI）锚定蛋白，用于选择产生高水平目标分泌蛋白的细胞。重组溶酶体酸性脂肪酶（LIPA）和α-半乳糖苷酶 A（GALA）与 GPI 附着信号序列和硒代半胱氨酸插入序列融合，在一个框内 UGA 密码子之后。在这些条件下，大多数重组蛋白被分泌到培养基中，但有些被发现是细胞表面的 GPI 锚定蛋白。当向培养基中供应亚硒酸钠时，GPI 锚定的 LIPA 和 GALA 的量增加。通过检测表面 GPI 锚定的 LIPA 来选择高表达细胞。然后通过敲除 GPI 生物合成基因 PIGK 去除 GPI 锚定蛋白，在这些细胞中，所有的 LIPA 都是分泌形式的。我们的系统为从大量细胞群体中分离高表达重组蛋白的细胞提供了一种很有前途的方法。
