Modular Integrated Secretory System Engineering in Pichia pastoris To Enhance G-Protein Coupled Receptor Expression.

TitleModular Integrated Secretory System Engineering in Pichia pastoris To Enhance G-Protein Coupled Receptor Expression.
Publication TypeJournal Article
Year of Publication2016
AuthorsClaes, K., K. Vandewalle, B. Laukens, T. Laeremans, O. Vosters, I. Langer, M. Parmentier, J. Steyaert, and N. Callewaert
JournalACS Synth Biol
Volume5
Issue10
Pagination1070-1075
Date Published2016 10 21
ISSN2161-5063
KeywordsAnimals, Camelids, New World, CHO Cells, Cricetulus, Gene Library, Genetic Engineering, Glycosylation, Metabolic Engineering, Pichia, Receptors, CXCR4, Receptors, G-Protein-Coupled, Recombinant Proteins, Single-Domain Antibodies, Unfolded Protein Response
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.

DOI10.1021/acssynbio.6b00032
Alternate JournalACS Synth Biol
PubMed ID27176489
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