Department of Antibody Discovery and Protein Engineering, MedImmune, LLC, One MedImmune Way, Gaithersburg, MD 20878, United States.
J Immunol Methods. 2013 Sep 30;395(1-2):45-53. doi: 10.1016/j.jim.2013.06.009. Epub 2013 Jul 4.
We describe herein a method to enable high throughput (HTP) screening of libraries of soluble proteins such as phage-derived clones of IgG, scFv-Fc, or other Fc-fusion proteins expressed in mammalian cells via adenovirus transduction. DNA fragments of antibody single chains (scFvs) and fragment antigen-binding (Fabs) from the positive clones of the third round of bacteriophage panning against a target antigen were batch reformatted into scFv-Fc or IgG in an oriP bearing entry vector and then recombined to an adenovirus vector through Gateway technology. The resulting antibody gene-containing adenovirus libraries were added to 96-well plates seeded with mammalian cells at a ratio of 0.7 infectious viral particles per well to establish clonality. Protocol optimization improved the expression of scFv-Fc and IgGs up to 100μg/mL in 96-well plates, which is sufficient for most antibody characterizations. In addition, 78% of the wells that were positive for protein expression contain only one sequence, indicating successful establishment of clonality in a majority of wells. We have established and optimized a mammalian expression system that produces soluble protein variants in a HTP manner. The system will facilitate developing multiple downstream screening methodologies.
我们在此描述了一种高通量 (HTP) 筛选方法，可用于筛选表达在哺乳动物细胞中的可溶性蛋白文库，如噬菌体衍生的 IgG 克隆、scFv-Fc 或其他 Fc 融合蛋白。针对靶抗原进行第三轮噬菌体淘选的阳性克隆中的抗体单链 (scFv) 和片段抗原结合 (Fab) 的 DNA 片段在 oriP 携带的-entry 载体中批量重新格式化为 scFv-Fc 或 IgG，然后通过 Gateway 技术重组到腺病毒载体中。将产生的含有抗体基因的腺病毒文库以每孔 0.7 个感染性病毒颗粒的比例添加到铺有哺乳动物细胞的 96 孔板中，以建立克隆性。通过优化方案，scFv-Fc 和 IgG 的表达量提高到 96 孔板中的 100μg/mL，足以进行大多数抗体表征。此外，78%的蛋白表达阳性孔仅含有一个序列，表明大多数孔中成功建立了克隆性。我们已经建立并优化了一种哺乳动物表达系统，可高通量生产可溶性蛋白变体。该系统将有助于开发多种下游筛选方法。
