GFP-based evaluation system of recombinant expression through the secretory pathway in insect cells and its application to the extracellular domains of class C GPCRs

Yuji Ashikawa, Makoto Ihara, Noriko Matsuura, Yuko Fukunaga, Yuko Kusakabe, Atsuko Yamashita

Research output: Contribution to journalArticle

14 Citations (Scopus)

Abstract

Applications of the GFP-fusion technique have greatly facilitated evaluations of the amounts and qualities of sample proteins used for structural analyses. In this study, we applied the GFP-based sample evaluation to secreted protein expression by insect cells. We verified that a GFP variant, GFPuv, retains proper folding and monodispersity within all expression spaces in Sf9 cells, such as the cytosol, organelles, and even the extracellular space after secretion, and thus can serve as a proper folding reporter for recombinant proteins. We then applied the GFPuv-based system to the extracellular domains of class C G-protein coupled receptors (GPCRs) and examined their localization, folding, and oligomerization upon insect cell expression. The extracellular domain of metabotropic glutamate receptor 1 (mGluR1) exhibited good secreted expression by Sf9 cells, and the secreted proteins formed dimer with a monodisperse hydrodynamic state favorable for crystallization, consistent with the results from previous successful structural analyses. In contrast, the extracellular domains of sweet/umami taste receptors (T1R) almost completely remained in the cell. Notably, the T1R and mGluR1 subfractions that remained in the cellular space showed polydisperse hydrodynamic states with large aggregated fractions, without forming dimers. These results indicated that the proper folding and oligomerization of the extracellular domains of the class C GPCR are achieved through the secretory pathway. Published by Wiley-Blackwell.

Original languageEnglish
Pages (from-to)1720-1734
Number of pages15
JournalProtein Science
Volume20
Issue number10
DOIs
Publication statusPublished - Oct 2011
Externally publishedYes

Fingerprint

Secretory Pathway
G-Protein-Coupled Receptors
Insects
Sf9 Cells
Oligomerization
Hydrodynamics
Dimers
Insect Proteins
Proteins
Extracellular Space
Crystallization
Recombinant Proteins
Organelles
Cytosol
Fusion reactions
metabotropic glutamate receptor type 1

Keywords

  • Class C GPCR
  • GFPuv
  • Green fluorescent protein
  • Precrystallization screening
  • Secretory pathway

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

Cite this

GFP-based evaluation system of recombinant expression through the secretory pathway in insect cells and its application to the extracellular domains of class C GPCRs. / Ashikawa, Yuji; Ihara, Makoto; Matsuura, Noriko; Fukunaga, Yuko; Kusakabe, Yuko; Yamashita, Atsuko.

In: Protein Science, Vol. 20, No. 10, 10.2011, p. 1720-1734.

Research output: Contribution to journalArticle

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