Selection of Antibodies to Transiently Expressed Membrane Proteins Using Phage Display

• Published in: Methods in molecular biology (Clifton, N.J.) Vol. 1827; p. 179
• Main Authors: Jones, Martina L, Mahler, Stephen M, Kumble, Sumukh
• Format: Journal Article
• Language: English
• Published: United States 2018
• Subjects: Transient transfection; Membrane protein; Cell-based biopanning; HEK293 cells; Green fluorescent protein; Cell sorting; CHO cells; Phage
• ISSN: 1940-6029
• DOI: 10.1007/978-1-4939-8648-4_10

Cell membrane proteins serve as attractive targets for biopharmaceutical development in addition to gauging their fundamental process in a biological system. Approximately 38% of the entire genome codes for plasma membrane proteins; however the discovery and development of antibody binders to such targets are a technical challenge. Methods to raise binders against such targets by cloning and expressing soluble extracellular regions have been met with limited success due to the loss of critical epitopes, with the resulting antibodies failing to bind to their target in its native conformation. This chapter outlines a "cell based biopanning" method in order to isolate antibodies against membrane proteins in their native conformation using transiently expressed, GFP-tagged target proteins. This method overcomes the limitations of non-specific binding of phage to the cells, abundance of irrelevant antigens on the cell surface, while retaining the native structure of the antigen on the cell surface.