Production of Recombinant Transmembrane Proteins from Mammalian Cells for Biochemical and Structural Analyses

Eukaryotic integral membrane proteins are key components of various biological processes. Because they are implicated in multiple diseases, it is important to understand their mechanism of action by elucidating their structure and function. Complex technical challenges associated with the generation of recombinant membrane proteins severely impair our ability to understand them using structural and biochemical methods. Here, we provide a detailed procedure to address and mitigate difficulties involved in the large-scale heterologous overexpression and purification of eukaryotic membrane proteins using HEK293S GnTi^- cells transduced with baculovirus. Two human proteins, hDHHC15 and hPORCN, are presented as examples, with step-by-step instructions for transient transfection and generation of baculoviruses, followed by overexpression and purification from HEK293S GnTi^- cells. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Small-scale protein expression in mammalian HEK293T cells Basic Protocol 2: Generation of baculovirus from Sf9 (insect) cells Alternate Protocol: Enumeration-free method for generating P₂ viral stock Support Protocol 1: Small-scale transduction of HEK293T cells with P₂ baculovirus Basic Protocol 3: Large-scale viral transduction of HEK293S GnTi^- cells Support Protocol 2: Large-scale membrane preparation from HEK293S GnTi^- cells Basic Protocol 4: Large-scale purification of membrane proteins from HEK293S GnTi^- cells.