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  Engineering of a proteolytically stable human β2-adrenergic receptor/maltose-binding protein fusion and production of the chimeric protein in Escherichia coli and baculovirus-infected insect cells

Hampe, W., Voss, R.-H., Haase, W., Boege, F., Michel, H., & Reiländer, H. (2000). Engineering of a proteolytically stable human β2-adrenergic receptor/maltose-binding protein fusion and production of the chimeric protein in Escherichia coli and baculovirus-infected insect cells. Journal of Biotechnology, 77(2-3), 219-234. doi:10.1016/s0168-1656(99)00216-3.

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 Creators:
Hampe, Wolfgang1, Author           
Voss, Ralf-Holger1, Author           
Haase, Winfried2, Author           
Boege, Fritz3, Author
Michel, Hartmut1, Author           
Reiländer, Helmut1, Author           
Affiliations:
1Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Max Planck Society, ou_2068290              
2Department of Structural Biology, Max Planck Institute of Biophysics, Max Planck Society, ou_2068291              
3Medizinische Poliklinik der Universität, 97070 Würzburg, Germany, ou_persistent22              

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Free keywords: Human β2-adrenergic receptor; Maltose-binding protein; Fusion protein; Proteolytic stability; Membrane protein purification
 Abstract: The hydrophobic human β2 adrenergic receptor was produced in fusion to the hydrophilic maltose-binding protein (MalE) in Escherichia coli. Photoaffinity labeling with the adrenergic ligand [125I]cyanopindolole-diazirine indicated that the majority of the protein was proteolyzed in the intergenic region between the fusion partners after production in E. coli. The simple and fast genetics of the bacterium enabled us to engineer a linker with an increased proteolytic stability. The fusion protein produced in E. coli was fully functional with respect to binding of adrenergic ligands and coupling to stimulatory GTP-binding protein. The production level with 3 pmol receptor fusion protein per mg membrane protein in a crude membrane preparation was significantly higher than those reported for other β2 adrenergic receptor constructs in E. coli. After solubilization with dodecanoyl sucrose, the fusion protein was purified to near homogeneity by affinity chromatography on immobilized Ni2+ ions (binding to a C-terminal His6-tag) and on crosslinked amylose (binding to the MalE). In order to achieve higher production levels, the fusion protein preceded by an insect signal peptide was produced in baculovirus-infected insect cells. As expected, the production level with about 17 pmol receptor per mg membrane protein was higher in the insect cells than in E. coli. The receptor fusion protein produced in the insect cells bound adrenergic ligands and activated heterotrimeric GTP-binding proteins with biochemical properties comparable to that of the unfused receptor.

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Language(s): eng - English
 Dates: 1999-10-041999-07-051999-10-132000-02-142000-02-17
 Publication Status: Issued
 Pages: 16
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Identifiers: DOI: 10.1016/s0168-1656(99)00216-3
PMID: 10682281
 Degree: -

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Title: Journal of Biotechnology
Source Genre: Journal
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Publ. Info: Elsevier
Pages: - Volume / Issue: 77 (2-3) Sequence Number: - Start / End Page: 219 - 234 Identifier: ISSN: 0168-1656
CoNE: https://pure.mpg.de/cone/journals/resource/954925484698