Coexpression of chaperonin GroEL/GroES markedly enhanced soluble and functional expression of recombinant human interferon-gamma in Escherichia coli.
Appl Microbiol Biotechnol
; 93(3): 1065-74, 2012 Feb.
Article
in En
| MEDLINE
| ID: mdl-21975693
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-ß-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.
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Biotechnology
/
Recombinant Proteins
/
Gene Expression Regulation, Bacterial
/
Interferon-gamma
/
Chaperonin 60
/
Chaperonin 10
/
Escherichia coli
Type of study:
Evaluation_studies
Limits:
Humans
Language:
En
Journal:
Appl Microbiol Biotechnol
Year:
2012
Document type:
Article