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Modular integrated secretory system engineering in Pichia pastoris to enhance G-protein coupled receptor expression

(2016) ACS SYNTHETIC BIOLOGY. 5(10). p.1070-1075
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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.
Keywords
Pichia pastoris, N-glycosylation, nanobodies, membrane protein expression, CXCR4, MEMBRANE-PROTEINS, CRYSTAL-STRUCTURE, RHODOPSIN, GENE, GPCR

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MLA
Claes, Katrien et al. “Modular Integrated Secretory System Engineering in Pichia Pastoris to Enhance G-protein Coupled Receptor Expression.” ACS SYNTHETIC BIOLOGY 5.10 (2016): 1070–1075. Print.
APA
Claes, Katrien, Vandewalle, K., Laukens, B., Laeremans, T., Vosters, O., Langer, I., Parmentier, M., et al. (2016). Modular integrated secretory system engineering in Pichia pastoris to enhance G-protein coupled receptor expression. ACS SYNTHETIC BIOLOGY, 5(10), 1070–1075.
Chicago author-date
Claes, Katrien, Kristof Vandewalle, Bram Laukens, Toon Laeremans, Olivier Vosters, Ingrid Langer, Marc Parmentier, Jan Steyaert, and Nico Callewaert. 2016. “Modular Integrated Secretory System Engineering in Pichia Pastoris to Enhance G-protein Coupled Receptor Expression.” Acs Synthetic Biology 5 (10): 1070–1075.
Chicago author-date (all authors)
Claes, Katrien, Kristof Vandewalle, Bram Laukens, Toon Laeremans, Olivier Vosters, Ingrid Langer, Marc Parmentier, Jan Steyaert, and Nico Callewaert. 2016. “Modular Integrated Secretory System Engineering in Pichia Pastoris to Enhance G-protein Coupled Receptor Expression.” Acs Synthetic Biology 5 (10): 1070–1075.
Vancouver
1.
Claes K, Vandewalle K, Laukens B, Laeremans T, Vosters O, Langer I, et al. Modular integrated secretory system engineering in Pichia pastoris to enhance G-protein coupled receptor expression. ACS SYNTHETIC BIOLOGY. 2016;5(10):1070–5.
IEEE
[1]
K. Claes et al., “Modular integrated secretory system engineering in Pichia pastoris to enhance G-protein coupled receptor expression,” ACS SYNTHETIC BIOLOGY, vol. 5, no. 10, pp. 1070–1075, 2016.
@article{8072509,
  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.}},
  author       = {{Claes, Katrien and Vandewalle, Kristof and Laukens, Bram and Laeremans, Toon and Vosters, Olivier and Langer, Ingrid and Parmentier, Marc and Steyaert, Jan and Callewaert, Nico}},
  issn         = {{2161-5063}},
  journal      = {{ACS SYNTHETIC BIOLOGY}},
  keywords     = {{Pichia pastoris,N-glycosylation,nanobodies,membrane protein expression,CXCR4,MEMBRANE-PROTEINS,CRYSTAL-STRUCTURE,RHODOPSIN,GENE,GPCR}},
  language     = {{eng}},
  number       = {{10}},
  pages        = {{1070--1075}},
  title        = {{Modular integrated secretory system engineering in Pichia pastoris to enhance G-protein coupled receptor expression}},
  url          = {{http://dx.doi.org/10.1021/acssynbio.6b00032}},
  volume       = {{5}},
  year         = {{2016}},
}

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