Xiao Xiaodong, Chen Yan, Mugabe Sheila, Gao Changshou, Tkaczyk Christine, Mazor Yariv, Pavlik Peter, Wu Herren, Dall'Acqua William, Chowdhury Partha Sarathi
Dept. of Antibody Discovery and Protein Engineering, MedImmune, LLC., Gaithersburg, MD, 20878, United States of America.
Dept. of Biopharmaceutical Development, MedImmune, LLC., Gaithersburg, MD, 20878, United States of America.
PLoS One. 2015 Oct 15;10(10):e0140691. doi: 10.1371/journal.pone.0140691. eCollection 2015.
High throughput screenings of single chain Fv (scFv) antibody phage display libraries are currently done as soluble scFvs produced in E.coli. Due to endotoxin contaminations from bacterial cells these preparations cannot be reliably used in mammalian cell based assays. The monovalent nature and lack of Fc in soluble scFvs prevent functional assays that are dependent on target cross linking and/or Fc functions. A convenient approach is to convert scFvs into scFv.Fc fusion proteins and express them in mammalian cell lines for screening. This approach is low throughput and is only taken after primary screening of monovalent scFvs that are expressed in bacteria. There is no platform at present that combines the benefits of both bacterial and mammalian expression system for screening phage library output. We have, therefore, developed a novel dual expression vector, called pSplice, which can be used to express scFv.Fc fusion proteins both in E.coli and mammalian cell lines. The hallmark of the vector is an engineered intron which houses the bacterial promoter and signal peptide for expression and secretion of scFv.Fc in E.coli. When the vector is transfected into a mammalian cell line, the intron is efficiently spliced out resulting in a functional operon for expression and secretion of the scFv.Fc fusion protein into the culture medium. By applying basic knowledge of mammalian introns and splisosome, we designed this vector to enable screening of phage libraries in a product like format. Like IgG, the scFv.Fc fusion protein is bi-valent for the antigen and possesses Fc effector functions. Expression in E.coli maintains the speed of the bacterial expression platform and is used to triage clones based on binding and other assays that are not sensitive to endotoxin. Triaged clones are then expressed in a mammalian cell line without the need for any additional cloning steps. Conditioned media from the mammalian cell line containing the fusion proteins are then used for different types of cell based assays. Thus this system retains the speed of the current screening system for phage libraries and adds additional functionality to it.
目前，单链Fv（scFv）抗体噬菌体展示文库的高通量筛选是通过在大肠杆菌中产生的可溶性scFv来完成的。由于细菌细胞产生的内毒素污染，这些制剂不能可靠地用于基于哺乳动物细胞的检测。可溶性scFv的单价性质和缺乏Fc结构域，使得依赖于靶标交联和/或Fc功能的功能检测无法进行。一种便捷的方法是将scFv转化为scFv.Fc融合蛋白，并在哺乳动物细胞系中表达以进行筛选。这种方法通量较低，且仅在对细菌中表达的单价scFv进行初步筛选后才采用。目前还没有一个平台能够结合细菌和哺乳动物表达系统的优势来筛选噬菌体文库的产物。因此，我们开发了一种新型双表达载体，称为pSplice，它可用于在大肠杆菌和哺乳动物细胞系中表达scFv.Fc融合蛋白。该载体的特点是一个工程化内含子，其中包含细菌启动子和信号肽，用于在大肠杆菌中表达和分泌scFv.Fc。当该载体转染到哺乳动物细胞系中时，内含子会被有效剪接掉，从而产生一个功能性操纵子，用于将scFv.Fc融合蛋白表达并分泌到培养基中。通过应用哺乳动物内含子和剪接体的基础知识，我们设计了这个载体，以便能够以类似产物的形式筛选噬菌体文库。与IgG一样，scFv.Fc融合蛋白对抗原具有双价性，并具有Fc效应功能。在大肠杆菌中的表达保持了细菌表达平台的速度，并用于基于结合和其他对内毒素不敏感的检测来筛选克隆。经过筛选的克隆随后无需任何额外的克隆步骤即可在哺乳动物细胞系中表达。然后，将含有融合蛋白的哺乳动物细胞系的条件培养基用于不同类型的基于细胞的检测。因此，该系统保留了当前噬菌体文库筛选系统的速度，并为其增加了额外的功能。
