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. 2015 Nov 20;5:16975. doi: 10.1038/srep16975

Figure 1. Synthesis of the interferon SIFα-IEP hybrid protein.

Figure 1

(A) Schematic diagram of “cDNA in frame fragment” library screening of interferon-enhanced peptides. Double-strand cDNAs from IFN-responding cells are sonicated. The gel-purified short fragments are ligated in frame with translation initiation code “ATG” of kanamycin. The “ATG-DCF-Kan+ colonies are selected by kanamycin and the “in-frame” DCFs are digested by BamH1/EcoRV and are ligated “in frame” to the C-terminus of IFN. The IFN-DCF lentiviral library is used to transfect 293T cells that carry the ISRE/copGFP/Puro+ reporter. The IFN-enhancer peptides (IEP) are recovered from cell clones that are resistant to puromycin and have the strong fluorescence of copGFP reporter. (B) Synthetic Interferon alpha (SIFα) vector. IEP: the consensus IFN-enhancer peptide. The positively charged amino acids are labeled in underlined red, where R = arginine, K = lysine. pCMV: CMV promoter; IFNα: wild type interferon alpha; SIFα: synthetic interferon alpha (IFNα-IEP); IEP vector: the control construct that contains only the IEP peptide; vector: vector control. (C) Predicted protein structures of IFNα (left panel) and the synthetic SIFα (right panel). The positively charged IEP peptide is shown in blue on the top of the IFNα structure. The putative structure of SIFα was predicted using the software on the website: http://zhanglab.ccmb.med.umich.edu.