Expression, refolding, and characterization of GFE peptide-fused human interferon-a2a in Escherichia coli

• Published in: Applied biochemistry and biotechnology Vol. 133; no. 2; pp. 149 - 162
• Main Authors: Yan, Zhen, Lu, Li, Shi, Jihong, Bao, Chunjie, Han, Wei, Wu, Yongjie, Zhang, Yingqi
• Format: Journal Article
• Language: English
• Published: 01.05.2006
• Subjects: Escherichia coli
• ISSN: 0273-2289; 1559-0291
• DOI: 10.1385/ABAB:133:2:149

Interferon-a2a (IFN-a2a) 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-a2a 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-a2a gene by recombinant DNA technique. Fusion protein rhIFN-a2a-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-a2a-GFE was found to be functionally active in terms of its antiviral activity for about 2.510 super(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-a2a 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-a2a 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-a2a to lungs. The results also showed that the IFN-a2a-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-a2a-GFE in liver may imply some significance in pharmacology and toxicology.