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Methods Mol Biol. 2018;1827:179-195. doi: 10.1007/978-1-4939-8648-4_10.

Selection of Antibodies to Transiently Expressed Membrane Proteins Using Phage Display.

Author information

1
ARC Training Centre for Biopharmaceutical Innovation, Australian Institute for Bioengineering and Nanotechnology (AIBN), The University of Queensland, St. Lucia, QLD, Australia. martina.jones@uq.edu.au.
2
ARC Training Centre for Biopharmaceutical Innovation, Australian Institute for Bioengineering and Nanotechnology (AIBN), The University of Queensland, St. Lucia, QLD, Australia.

Abstract

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.

KEYWORDS:

CHO cells; Cell sorting; Cell-based biopanning; Green fluorescent protein; HEK293 cells; Membrane protein; Phage; Transient transfection

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