Structural characterization of recombinant human CD81 produced in Pichia pastoris

Jamshad, Mohammed, Rajesh, Sundaresan, Stamataki, Zania, McKeating, Jane A, Dafforn, Timothy, Overduin, Michael and Bill, Roslyn M. (2008). Structural characterization of recombinant human CD81 produced in Pichia pastoris. Protein Expression and Purification, 57 (2), pp. 206-216.

Abstract

Human CD81 (hCD81) protein has been recombinantly produced in the methylotrophic yeast Pichia pastoris. The purified protein, produced at a yield of 1.75 mg/L of culture, was shown to interact with Hepatitis C virus E2 glycoprotein. Immunofluorescent and flow cytometric staining of P. pastoris protoplasts with monoclonal antibodies specific for the second extracellular loop (EC2) of hCD81 confirmed the antigenicity of the recombinant molecule. Full-length hCD81 was solubilized with an array of detergents and subsequently characterized using circular dichroism (CD) and analytical ultracentrifugation. These biophysical techniques confirmed that the protein solution comprises a homogenous species possessing a highly-defined alpha-helical secondary structure. The predicted alpha-helical content of the protein from CD analysis (77.1%) fits remarkably well with what would be expected (75.2%) from knowledge of the protein sequence together with the data from the crystal structure of the second extracellular loop. This study represents the first biophysical characterization of a full-length recombinant tetraspanin, and opens the way for structure-activity analyses of this ubiquitous family of transmembrane proteins.

Publication DOI: https://doi.org/10.1016/j.pep.2007.10.013
Divisions: Life & Health Sciences > Biosciences
Life & Health Sciences
Life & Health Sciences > Cellular and Molecular Biomedicine
Additional Information: © 2007 Elsevier Inc. Open access under CC BY license
Uncontrolled Keywords: membrane proteins,protein production,yeast,circular dichroism,analytical ultracentrifugation,Biochemistry
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Related URLs: http://www.scop ... tnerID=8YFLogxK (Scopus URL)
Published Date: 2008-02
Authors: Jamshad, Mohammed
Rajesh, Sundaresan
Stamataki, Zania
McKeating, Jane A
Dafforn, Timothy
Overduin, Michael
Bill, Roslyn M. ( 0000-0003-1331-0852)

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