Expression, refolding, and characterization of GFE peptide-fused human interferon-alpha2a in Escherichia coli.
Interferon-alpha2a (IFN-alpha2a) 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-alpha2a 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-alphaa gene by recombinant DNA technique. Fusion protein rhIFN-alpha2a-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-alpha2a-GFE was found to be functionally active in terms of its antiviral activity for about 2.5 x 108 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-alpha2a 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-alphaa 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-alpha2a to lungs. The results also showed that the IFN-alpha2a-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-alpha2a-GFE in liver may imply some significance in pharmacology and toxicology.
Yan
Zhen
Z
Biotechnology Center of Fourth Military Medical University, Chang Le Xi Lu #17, Xi'an 710032, Shaanxi, People's Republic of China.
Lu
Li
L
Shi
Jihong
J
Bao
Chunjie
C
Han
Wei
W
Wu
Yongjie
Y
Zhang
Yingqi
Y
D000818:Animals; D000998:Antiviral Agents / Q000138:chemical synthesis / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D005786:Gene Expression Regulation; D006801:Humans; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D007668:Kidney / Q000737:chemistry / Q000378:metabolism; D008168:Lung / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D009842:Oligopeptides / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D011485:Protein Binding; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins
D000998:Antiviral Agents; C118896:GFE-1 peptide; D000077190:Interferon alpha-2; D016898:Interferon-alpha; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins