The human ET(B) endothelin receptor heterologously produced in the 
methylotrophic yeast Pichia pastoris shows high-affinity binding and induction 
of stacked membranes

Schiller, H.; Haase, Winfried; Molsberger, E.; Janssen, P.; Michel, Hartmut; Reiländer, Helmut

アイテム詳細

登録内容を編集ファイル形式で保存

一時保存へ追加

タグ情報を表示リリース履歴を表示詳細要約

公開

学術論文

The human ET(B) endothelin receptor heterologously produced in the methylotrophic yeast Pichia pastoris shows high-affinity binding and induction of stacked membranes

MPS-Authors

/persons/resource/persons137691

Haase, Winfried
Department of Structural Biology, Max Planck Institute of Biophysics, Max Planck Society;

/persons/resource/persons137800

Michel, Hartmut
Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Max Planck Society;

/persons/resource/persons137849

Reiländer, Helmut
Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Max Planck Society;

External Resource

There are no locators available

Fulltext (restricted access)

There are currently no full texts shared for your IP range.

フルテキスト (公開)

公開されているフルテキストはありません

付随資料 (公開)

There is no public supplementary material available

引用

Schiller, H., Haase, W., Molsberger, E., Janssen, P., Michel, H., & Reiländer, H. (2000). The human ET(B) endothelin receptor heterologously produced in the methylotrophic yeast Pichia pastoris shows high-affinity binding and induction of stacked membranes. Receptors and Channels, 7(2), 93-107.

引用: https://hdl.handle.net/21.11116/0000-000D-4A37-3

要旨

The human endothelin B receptor (ET(B) receptor) was produced in the methylotrophic yeast Pichia pastoris under transcriptional control of the highly inducible alcohol oxidase 1 (AOX1) gene promoter. In the expression plasmids pPIC9KFlagET(B)Bio and pPIC9KFlag deltaGPET(B)Bio the ET(B) receptor coding region was fused in frame to the Saccharomyces cerevisiae alpha-factor prepropeptide and the FLAG-tag. In both constructs, the receptor was also fused to a biotinylation domain. Additionally, in pPIC9KFlag deltaGPET(B)Bio the putative N-glycosylation site and a protease site have been deleted by site directed mutagenesis. Crude membranes prepared from recombinant P. pastoris revealed specific and saturable binding of [125I]ET-1 with a K(D) of about 42 pM. Receptor levels of 60 pmol/mg and 35 pmol/mg for the Flag deltaGPET(B)Bio and the FlagET(B)Bio construct, respectively, were determined. The pharmacological profile for ET-1, ET-2 and ET-3 were as expected for a subtype B endothelin (ET) receptor. Immunoblot analysis showed an apparent molecular mass of 55 kDa for the Kex2-processed and about 74 kDa for the Kex2-unprocessed receptor. Contrary to the Flag deltaGPET(B)Bio construct, the FlagET(B)Bio construct was not correctly processed by the internal Kex2 endopeptidase. As was detected by ultrastructural analysis of recombinant yeast cells, high-level production of the receptor resulted in the formation of stacked membranes.