Cloning and Overexpressing Membrane Proteins Using Pichia pastoris (Komagataella phaffii)

Understanding the structure and function of key proteins located within biological membranes is essential for fundamental knowledge and therapeutic applications. Robust cell systems allowing their actual overexpression are required, among which stands the methylotrophic yeast Pichia pastoris. This system proves highly efficient in producing many eukaryotic membrane proteins of various functions and structures at levels and quality compatible with their subsequent isolation and molecular investigation. This article describes a set of basic guidelines and directions to clone and select recombinant P. pastoris clones overexpressing eukaryotic membrane proteins. Illustrative results obtained for a panel of mammalian membrane proteins are presented, and hints are given on a series of experimental parameters that may substantially improve the amount and/or the functionality of the expressed proteins. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Designing and cloning a P. pastoris expression vector Basic Protocol 2: Integrative transformation of P. pastoris and selection of recombinant clones Basic Protocol 3: Culturing transformed P. pastoris for membrane protein expression Basic Protocol 4: Yeast cell lysis and membrane preparation Basic Protocol 5: Immunodetection of expressed membrane proteins: western blot Alternate Protocol 1: Immunodetection of expressed membrane proteins: dot blot Alternate Protocol 2: Immunodetection of expressed membrane proteins: yeastern blot Basic Protocol 6: Activity assay: ligand‐binding analysis of an expressed GPCR