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Overcoming Challenges of Hepatitis C Virus Envelope Glycoprotein Production in Mammalian Cells

  • Samantha A. Yost
  • Jillian Whidby
  • Abdul G. Khan
  • Yuanyuan Wang
  • Joseph Marcotrigiano
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1911)

Abstract

Posttranslational modifications (PTMs) are often required for proper folding and physiological function of proteins, including the envelope glycoproteins 1 and 2 (E1 and E2) of hepatitis C virus (HCV). Commonly used expression systems such as bacteria, yeast, and baculovirus produce soluble HCV E1 and E2 at very low yields, as the cellular environment and molecular machinery necessary for PTMs may be suboptimal or missing. Here, we describe an expression system for HCV E2 ectodomain (eE2) with 11 N-linked glycans and eight disulfide bonds, which combines lentivirus transduction of mammalian cells and a continuous growth, adherent cell bioreactor. It is environmentally friendly, as well as cost- and time-efficient compared to other methods of recombinant protein expression in mammalian systems with final yields of eE2 approaching 60 mg/L of cell culture supernatant. eE2 produced by this system is amenable to a variety of biophysical studies, including structural determination by X-ray crystallography. Considering the ease of use and flexibility, this method can be applied to express an array of difficult target proteins in a variety of mammalian cell lines.

Key words

E2 Lentivirus Protein expression Purification HCV Hepatitis C virus Glycoproteins Posttranslational modification 

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Samantha A. Yost
    • 1
  • Jillian Whidby
    • 1
  • Abdul G. Khan
    • 1
  • Yuanyuan Wang
    • 1
    • 2
  • Joseph Marcotrigiano
    • 2
  1. 1.Department of Chemistry and Chemical Biology, Center for Advanced Biotechnology and MedicineRutgers UniversityPiscatawayUSA
  2. 2.Laboratory of Infectious DiseasesNational Institute of Allergy and Infectious Diseases, National Institute of HealthBethesdaUSA

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