title: Modular Integrated Secretory System Engineering in Pichia pastoris To Enhance G-Protein Coupled Receptor Expression.
abstract: Membrane protein research is still hampered by the generally very low levels at which these proteins are naturally expressed, necessitating heterologous expression. Protein degradation, folding problems, and undesired post-translational modifications often occur, together resulting in low expression levels of heterogeneous protein products that are unsuitable for structural studies. We here demonstrate how the integration of multiple engineering modules in Pichia pastoris can be used to increase both the quality and the quantity of overexpressed integral membrane proteins, with the human CXCR4 G-protein coupled receptor as an example. The combination of reduced proteolysis, enhanced ER folding capacity, GlycoDelete-based N-Glycan trimming, and nanobody-based fold stabilization improved the expression of this GPCR in P. pastoris from a low expression level of a heterogeneously glycosylated, proteolyzed product to substantial quantities (2-3 mg/L shake flask culture) of a nonproteolyzed, homogeneously glycosylated proteoform. We expect that this set of tools will contribute to successful expression of more membrane proteins in a quantity and quality suitable for functional and structural studies.
authors.affiliation: Unit for Medical Biotechnology, Medical Biotechnology Center, VIB , Technologiepark 927, 9052 Ghent, Belgium.|Laboratory for Protein Biochemistry and Biomolecular Engineering, Department of Biochemistry and Microbiology, Ghent University , K.L.-Ledeganckstraat 35, 9000 Ghent, Belgium.
authors.forename: Katrien
authors.initials: K
authors.lastname: Claes
authors.affiliation: Unit for Medical Biotechnology, Medical Biotechnology Center, VIB , Technologiepark 927, 9052 Ghent, Belgium.|Laboratory for Protein Biochemistry and Biomolecular Engineering, Department of Biochemistry and Microbiology, Ghent University , K.L.-Ledeganckstraat 35, 9000 Ghent, Belgium.
authors.forename: Kristof
authors.initials: K
authors.lastname: Vandewalle
authors.affiliation: Unit for Medical Biotechnology, Medical Biotechnology Center, VIB , Technologiepark 927, 9052 Ghent, Belgium.|Laboratory for Protein Biochemistry and Biomolecular Engineering, Department of Biochemistry and Microbiology, Ghent University , K.L.-Ledeganckstraat 35, 9000 Ghent, Belgium.
authors.forename: Bram
authors.initials: B
authors.lastname: Laukens
authors.affiliation: Structural Biology Research Center, VIB , Pleinlaan 2, 1050 Brussels, Belgium.|Structural Biology Brussels, Vrije Universiteit Brussel , Pleinlaan 2, 1050 Brussels, Belgium.
authors.forename: Toon
authors.initials: T
authors.lastname: Laeremans
authors.affiliation: Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire (IRIBHM) , Campus Erasme, 808 Route de Lennik, B-1070 Brussels, Belgium.
authors.forename: Olivier
authors.initials: O
authors.lastname: Vosters
authors.affiliation: Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire (IRIBHM) , Campus Erasme, 808 Route de Lennik, B-1070 Brussels, Belgium.
authors.forename: Ingrid
authors.initials: I
authors.lastname: Langer
authors.affiliation: Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire (IRIBHM) , Campus Erasme, 808 Route de Lennik, B-1070 Brussels, Belgium.|Welbio, Université Libre de Bruxelles (U.L.B.) , Campus Erasme, 808 Route de Lennik, B-1070 Brussels, Belgium.
authors.forename: Marc
authors.initials: M
authors.lastname: Parmentier
authors.affiliation: Structural Biology Research Center, VIB , Pleinlaan 2, 1050 Brussels, Belgium.|Structural Biology Brussels, Vrije Universiteit Brussel , Pleinlaan 2, 1050 Brussels, Belgium.
authors.forename: Jan
authors.initials: J
authors.lastname: Steyaert
authors.affiliation: Unit for Medical Biotechnology, Medical Biotechnology Center, VIB , Technologiepark 927, 9052 Ghent, Belgium.|Laboratory for Protein Biochemistry and Biomolecular Engineering, Department of Biochemistry and Microbiology, Ghent University , K.L.-Ledeganckstraat 35, 9000 Ghent, Belgium.
authors.forename: Nico
authors.initials: N
authors.lastname: Callewaert
mesh_terms: D000818:Animals; D016466:CHO Cells; D002161:Camelids, New World; D003412:Cricetulus; D015723:Gene Library; D005818:Genetic Engineering / Q000379:methods*; D006031:Glycosylation; D060847:Metabolic Engineering / Q000379:methods; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D019718:Receptors, CXCR4 / Q000235:genetics / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics* / Q000378:metabolism; D061905:Single-Domain Antibodies / Q000378:metabolism; D056811:Unfolded Protein Response / Q000235:genetics
chemical_list: C527276:CXCR4 protein, human; D019718:Receptors, CXCR4; D043562:Receptors, G-Protein-Coupled; D011994:Recombinant Proteins; D061905:Single-Domain Antibodies
keywords: CXCR4; N-glycosylation; Pichia pastoris; membrane protein expression; nanobodies