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.

大多数生物药物蛋白质是在哺乳动物细胞中产生的，因为它们具有蛋白质折叠，组装和翻译后修饰的有利能力。为了满足对用于临床目的和研究的这些蛋白质的日益增长的需求，提高蛋白质生产率的传统方法包括基因扩增，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。在这项研究中，我们建立了糖基磷脂酰肌醇（GPI）锚定蛋白和硒代半胱氨酸（GPS）系统，以选择产生高水平目标分泌蛋白的细胞。在框内UGA密码子后，将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。在这些条件下，大多数重组蛋白被分泌到培养基中，但是发现其中一些是细胞表面上GPI锚定的蛋白。当将亚硒酸钠供应到培养基中时，GPI锚定的LIPA和GALA的量增加。通过检测表面GPI锚定的LIPA选择高表达的细胞。然后通过敲除GPI生物合成基因来消除GPI锚定的蛋白质 当将亚硒酸钠供应到培养基中时，GPI锚定的LIPA和GALA的量增加。通过检测表面GPI锚定的LIPA选择高表达的细胞。然后通过敲除GPI生物合成基因来消除GPI锚定的蛋白质 当将亚硒酸钠供应到培养基中时，GPI锚定的LIPA和GALA的量增加。通过检测表面GPI锚定的LIPA选择高表达的细胞。然后通过敲除GPI生物合成基因来消除GPI锚定的蛋白质PIGK，在这些细胞中，所有LIPA是分泌形式。我们的系统提供了一种从大细胞群体中分离高表达重组蛋白的细胞的有前途的方法。