Membrane proteins play important roles in many biological processes and are a major drug target. However, only a limited number of structures of eukaryotic membrane proteins have been determined so far. Besides the challenges in crystallizing these proteins, one of the main bottlenecks in structure determination is their recombinant expression. The mammalian HEK293 cell line provides a natural environment for expression of eukaryotic membrane proteins but optimization of transfection and cultivation time is often necessary to yield amounts of protein suitable for structural studies.Here we describe a detailed protocol for expression and purification of membrane proteins from HEK293 cells with an example of the human peptide transporter, PepT2. In the first part, we focus on the expression optimization by changing transfection protocol and cultivation time. In the second part, we describe a robust protocol for large-scale expression and purification of membrane proteins based on affinity chromatography and gel filtration.
Keywords: HEK293 cell suspension culture; Membrane protein; Protein expression; Protein purification; Transient transfection.