Applied Microbiology and Biotechnology

, Volume 93, Issue 3, pp 1065–1074 | Cite as

Coexpression of chaperonin GroEL/GroES markedly enhanced soluble and functional expression of recombinant human interferon-gamma in Escherichia coli

  • Xiao Yan
  • Sheng Hu
  • Yi-Xin GuanEmail author
  • Shan-Jing Yao
Biotechnological products and process engineering


Recombinant human interferon-gamma (rhIFN-γ) is a protein of great potential for clinical therapy due to its multiple biological activities. However, overexpressing rhIFN-γ in Escherichia coli was found to accumulate as cytoplasmic inclusion bodies. In this work, a system for soluble and active expression of rhIFN-γ was constructed by coexpressing chaperonin GroEL/GroES in E. coli. The rhIFN-γ gene was fused to a pET-28a expression vector, and rhIFN-γ was partially expressed as the soluble form following coexpression with a second vector producing chaperonin GroEL/GroES. The fermentation of recombinant E. coli harboring rhIFN-γ and GroEL/GroES plasmids was investigated, and the optimized conditions were as follows: culture temperature of 25°C, incubation time of 8 h, isopropyl-β-d-thio-galactoside concentration of 0.2 mM, and l-arabinose concentration of 0.5 g/L. As a result, the expression level of rhIFN-γ was improved accordingly by 2.2-fold than the control, while a significantly positive correlation was also found between the ratio of supernatant to precipitate of rhIFN-γ and the amount of chaperonin. Circular dichroism spectra, fluorescence spectra, size exclusion chromatography, and chemical cross-linking method were applied to characterize rhIFN-γ, indicating that the three-dimensional structure of rhIFN-γ was identical to that of the native rhIFN-γ. The enzyme-linked immunosorbent assay for active rhIFN-γ quantification showed that coexpression yielded 72.91 mg rhIFN-γ per liter fermentation broth. Finally, protein–protein interactions between rhIFN-γ and chaperonin were analyzed using the yeast two-hybrid system, which provided the direct evidence that chaperonin GroEL/GroES interacted with rhIFN-γ to increase the soluble expression and presented the potential in producing efficiently recombinant proteins.


Recombinant human interferon-gamma GroEL/GroES coexpression system Soluble expression Protein folding Protein–protein interactions 



This work was financially supported by the National Natural Science Foundation of China (no. 20876138).


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Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Xiao Yan
    • 1
  • Sheng Hu
    • 2
  • Yi-Xin Guan
    • 1
    Email author
  • Shan-Jing Yao
    • 1
  1. 1.Department of Chemical and Biological EngineeringZhejiang UniversityHangzhouPeople’s Republic of China
  2. 2.School of Biotechnology and Chemical Engineering, Ningbo Institute of TechnologyZhejiang UniversityNingboPeople’s Republic of China

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