|Title||Modular Integrated Secretory System Engineering in Pichia pastoris To Enhance G-Protein Coupled Receptor Expression.|
|Publication Type||Journal Article|
|Year of Publication||2016|
|Authors||Claes, K., K. Vandewalle, B. Laukens, T. Laeremans, O. Vosters, I. Langer, M. Parmentier, J. Steyaert, and N. Callewaert|
|Journal||ACS Synth Biol|
|Date Published||2016 10 21|
|Keywords||Animals, 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|
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.
|Alternate Journal||ACS Synth Biol|
Modular Integrated Secretory System Engineering in Pichia pastoris To Enhance G-Protein Coupled Receptor Expression.