Applied Microbiology and Biotechnology

, Volume 90, Issue 1, pp 181–191

A rational design for hepatitis B virus X protein refolding and bioprocess development guided by second virial coefficient studies

  • Anindya Basu
  • Wei Ning Chen
  • Susanna Su Jan Leong
Biotechnologically Relevant Enzymes and Proteins

DOI: 10.1007/s00253-010-3058-5

Cite this article as:
Basu, A., Chen, W.N. & Leong, S.S.J. Appl Microbiol Biotechnol (2011) 90: 181. doi:10.1007/s00253-010-3058-5

Abstract

The hepatitis B virus X (HBx) protein is well known for its role in hepatitis B virus infection that often leads to hepatocellular carcinoma. Despite the clinical importance of HBx, there is little progress in anti-HBx drug development strategies due to shortage of HBx from native sources. Consistent expression of HBx as insoluble inclusion bodies within various expression systems has largely hindered HBx manufacturing via economical biosynthesis routes. Confronted by this roadblock, this study aims to quantitatively understand HBx protein behaviour in solution that will guide the rational development of a refolding-based bioprocess for HBx production. Second virial coefficient (SVC) measurements were employed to study the effects of varying physicochemical parameters on HBx intermolecular protein interaction. The SVC results suggest that covalent HBx aggregates play a key role in protein destabilisation during refolding. The use of an SVC-optimised refolding environment yielded bioactive and soluble HBx proteins from the denatured–reduced inclusion body state. This study provides new knowledge on HBx solubility behaviour in vitro, which is important in structure–function elucidation behaviour of this hydrophobic protein. Importantly, a rational refolding-based Escherichia coli bioprocess that can deliver purified and soluble HBx at large scale is successfully developed, which opens the way for rapid preparation of soluble HBx for further clinical and characterisation studies.

Keywords

Hepatitis B virus X proteinSecond virial coefficientSolubilityBioprocessRefolding

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Anindya Basu
    • 1
  • Wei Ning Chen
    • 1
  • Susanna Su Jan Leong
    • 1
  1. 1.School of Chemical and Biomedical EngineeringNanyang Technological UniversityNanyangSingapore