title: Coexpression of chaperonin GroEL/GroES markedly enhanced soluble and functional expression of recombinant human interferon-gamma in Escherichia coli.
abstract: 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 crosslinking 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.
authors: Department of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, People's Republic of China.
authors: Xiao
authors: X
authors: Yan
authors: Sheng
authors: S
authors: Hu
authors: Yi-Xin
authors: YX
authors: Guan
authors: Shan-Jing
authors: SJ
authors: Yao
mesh_terms: D001709:Biotechnology / Q000379:methods*; D018835:Chaperonin 10 / Q000378:metabolism*; D018834:Chaperonin 60 / Q000378:metabolism*; D003470:Culture Media; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D005285:Fermentation; D015964:Gene Expression Regulation, Bacterial; D006801:Humans; D007371:Interferon-gamma / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D017510:Protein Folding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D012995:Solubility; D020798:Two-Hybrid System Techniques
chemical_list: D018835:Chaperonin 10; D018834:Chaperonin 60; D003470:Culture Media; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D007371:Interferon-gamma