Structural characterization of recombinant human CD81 produced in Pichia pastoris

Mohammed Jamshad, Sundaresan Rajesh, Zania Stamataki, Jane A McKeating, Timothy Dafforn, Michael Overduin, Roslyn M. Bill

Research output: Contribution to journalArticle

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.
Original languageEnglish
Pages (from-to)206-216
Number of pages11
JournalProtein Expression and Purification
Volume57
Issue number2
DOIs
Publication statusPublished - Feb 2008

Fingerprint

Pichia
Circular Dichroism
Proteins
Protoplasts
Ultracentrifugation
Detergents
Yeast
Yeasts
Crystal structure
Monoclonal Antibodies
Staining and Labeling
Molecules

Bibliographical note

© 2007 Elsevier Inc. Open access under CC BY license

Keywords

  • membrane proteins
  • protein production
  • yeast
  • circular dichroism
  • analytical ultracentrifugation

Cite this

Jamshad, Mohammed ; Rajesh, Sundaresan ; Stamataki, Zania ; McKeating, Jane A ; Dafforn, Timothy ; Overduin, Michael ; Bill, Roslyn M. / Structural characterization of recombinant human CD81 produced in Pichia pastoris. In: Protein Expression and Purification. 2008 ; Vol. 57, No. 2. pp. 206-216.
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Jamshad, M, Rajesh, S, Stamataki, Z, McKeating, JA, Dafforn, T, Overduin, M & Bill, RM 2008, 'Structural characterization of recombinant human CD81 produced in Pichia pastoris', Protein Expression and Purification, vol. 57, no. 2, pp. 206-216. https://doi.org/10.1016/j.pep.2007.10.013

Structural characterization of recombinant human CD81 produced in Pichia pastoris. / Jamshad, Mohammed; Rajesh, Sundaresan; Stamataki, Zania; McKeating, Jane A; Dafforn, Timothy; Overduin, Michael; Bill, Roslyn M.

In: Protein Expression and Purification, Vol. 57, No. 2, 02.2008, p. 206-216.

Research output: Contribution to journalArticle

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Jamshad M, Rajesh S, Stamataki Z, McKeating JA, Dafforn T, Overduin M et al. Structural characterization of recombinant human CD81 produced in Pichia pastoris. Protein Expression and Purification. 2008 Feb;57(2):206-216. https://doi.org/10.1016/j.pep.2007.10.013