Coexpression of chaperonin GroEL/GroES markedly enhanced soluble and functional expression of recombinant human interferon-gamma in <Emphasis Type="Italic">Escherichia coli</Emphasis> |
| |
Authors: | Xiao?Yan Sheng?Hu Email author" target="_blank">Yi-Xin?GuanEmail author Shan-Jing?Yao |
| |
Institution: | (1) Department of Chemical and Biological Engineering, Zhejiang University, Hangzhou, 310027, People’s Republic of China;(2) School of Biotechnology and Chemical Engineering, Ningbo Institute of Technology, Zhejiang University, Ningbo, 315100, People’s Republic of China; |
| |
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 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. |
| |
Keywords: | |
本文献已被 PubMed SpringerLink 等数据库收录! |
|