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  Solubilization and purification of the human ETB endothelin receptor produced by high-level fermentation in Pichia pastoris

Schiller, H., Molsberger, E., Janssen, P., Michel, H., & Reiländer, H. (2001). Solubilization and purification of the human ETB endothelin receptor produced by high-level fermentation in Pichia pastoris. Receptors and Channels, 7(6), 453-469.

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 Creators:
Schiller, Hilmar1, Author              
Molsberger, Eva1, Author              
Janssen , P.1, Author
Michel, Hartmut1, Author              
Reiländer, Helmut1, Author              
Affiliations:
1Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Max Planck Society, ou_2068290              

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 Abstract: In the present report, the successful solubilization and purification of the ETB receptor heterologously produced in the methylotrophic yeast P. pastoris is described for the first time. In comparison to the baculovirus system where successful production, solubilization and purification have already been reported, handling and up-scaling of recombinant P. pastoris cells was much easier and less time consuming. Recombinant P. pastoris clones producing two different ETB receptor constructs were grown in a fermenter to a density of about 360 g/l. After induction with methanol, a production level of maximally 45 pmol/mg was obtained, a value which is in the range of that reported for baculovirus-infected insect cells. A method for the large-scale preparation of membranes was established. Solubilization of the recombinant ETB receptor was achieved with the detergent n-dodecyl-/beta-D-maltopyranoside. The stability of the solubilized and ligand-bound receptor was examined in detail. Subsequently, two purification methods for two different receptor constructs were tested and a large-scale procedure for isolation of recombinant receptor was established. In general, the purification methods described herein will be adaptable to other G protein-coupled receptors heterologously produced in heterologous expression systems including P. pastoris.

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Language(s): eng - English
 Dates: 2001-01-01
 Publication Status: Published in print
 Pages: 17
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Identifiers: PMID: 11918348
 Degree: -

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Title: Receptors and Channels
Source Genre: Journal
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Publ. Info: Yverdon, Switzerland : Harwood Academic Publishers
Pages: - Volume / Issue: 7 (6) Sequence Number: - Start / End Page: 453 - 469 Identifier: ISSN: 1060-6823
CoNE: https://pure.mpg.de/cone/journals/resource/954925597599