Expression, refolding, and characterization of GFE peptide-fused human interferon-[alpha]2a in Escherichia coli

• Published in: Applied biochemistry and biotechnology Vol. 133; no. 2; p. 149
• Main Authors: Yan, Zhen, Lu, Li, Shi, Jihong, Bao, Chunjie, Han, Wei, Wu, Yongjie, Zhang, Yingqi
• Format: Journal Article
• Language: English
• Published: Totowa Springer Nature B.V 01.05.2006
• Subjects: Dialysis; E coli; Gene expression; Kidneys; Peptides; Pharmacology; Proteins; RNA polymerase; Studies; Toxicology
• DOI: 10.1385/ABAB:133:2:149
• ISSN: 0273-2289

Interferon-α2a (IFN-α2a) has been used for the treatment of various viral infections and cancers for many years. However some untolerable side effects have limited its application in some aspects. To evaluate whether or not an oligopeptide containing GFE motif can home human IFN-α2a to specific tissues, a fusion gene was constructed by fusing the coding sequence of GFE peptide (CGFECVRQCPERC), which was screened from phage display peptide library, to the 3' end of human IFN-α2a gene by recombinant DNA technique. Fusion protein rhIFN-α2a-GFE was expressed in Escherichia coli as inclusion bodies using a T7 RNA polymerase expression system, pET-22b, refolded through dialysis and purified to homogeneity to >95% of purity by affinity chromatography. Characterization by sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotting demonstrated the authenticity of the fusion protein. Purified rhIFN-α2a-GFE was found to be functionally active in terms of its antiviral activity for about 2.5×10^sup 8^ IU/mg in vitro. Yields of the purified fusion protein were about 200 mg/L of culture medium. Tissue distribution assay in mouse showed that at 30 min IFN-α2a could be enriched sevenfold higher in lung in the targeted IFN group of mice than in the standard IFN group of mice, and last for a long time. At 1 h, IFN-α2a in the targeted IFN group was still 4.02-fold higher than that in the standard group. This confirmed that GFE peptide has the ability to selectively deliver its fusion partner IFN-α2a to lungs. The results also showed that the IFN-α2a-GFE could be specifically enriched in kidney and liver. Its distribution in kidney was concordant with the finding of GFE receptor, MDP, in kidney. However, the IFN-α2a-GFE in liver may imply some significance in pharmacology and toxicology.[PUBLICATION ABSTRACT]