Abstract
BackgroundIsopropyl-β-D-1-thiolgalactopyranoside (IPTG)-inducible expression of recombinant proteins in E. coli is commonly used and effective. Nevertheless, unintended induction was encountered as a problem when using these bacterial expression systems, generating cultures that give reduced or variable protein yields. Auto-induction allows for production of much higher target protein yield and cell mass than conventional procedures using induction with IPTG without monitoring cell growth then adding IPTG at the appropriate cell density. This method involves special media recipes that promote growth to high density and automatically induce expression of target protein from T7 promoter. Consensus interferon is a synthetic artificially engineered interferon having an amino acid sequence that is a rough average of the sequences of all natural human alpha interferon subtypes and has greater potency than other interferons even the pegylated versions. The purpose of this study was high-level expression of human consensus interferon-alpha (cIFN-α) in E. coli using an auto-induction protocol. The cIFN-α gene was cloned into pET101/D-TOPO expression vector under the T7 promoter transcriptional regulation. Expression was optimized with respect to temperature and length of incubation in shake flask cultures. The antiviral potency and anticancer activity of cIFN-α were evaluated in comparison to IFN-α2a.ResultsThe expressed cIFN-α protein in auto-induction T7 system was found mostly in soluble fraction of the cell lysate (about 70% of yield in total cell lysate) after lowering incubation temperature to 25°C or 30°C. Protein expression was maximal after 24 h incubation at 25°C or 30°C. After purification via single-step chromatography using DEAE-Sepharose, the yield was 270 mg/L in shake flask E. coli cultures which is much higher than IPTG-inducible T7 expression system and other systems according to available data. The synthesized cIFN-α was biologically active as confirmed by its anticancer and antiviral effects and was significantly more potent than IFN-α2a.ConclusionsThe auto-induction process was reliable and convenient for production of cIFN-α protein in E. coli, and can be adapted for large-scale therapeutic protein production.
Highlights
Isopropyl-β-D-1-thiolgalactopyranoside (IPTG)-inducible expression of recombinant proteins in E. coli is commonly used and effective
Plasmid construction for auto-induction expression The blunt-ended PCR product of the synthetic gene coding for Human consensus interferon-alpha (cIFN-α) was directionally cloned into pET101/DTOPO vector by adding four bases (CACC) to the forward primer
The constructed pET-cIFNα plasmid was transformed into E. coli BL21 (DE3) for cIFN-α auto-induction expression under the control of the T7 promoter
Summary
Isopropyl-β-D-1-thiolgalactopyranoside (IPTG)-inducible expression of recombinant proteins in E. coli is commonly used and effective. Interferons (IFNs) are cytokines secreted by vertebrates’ cells when stimulated by viruses and several other agents [1,2] This family of glycoproteins exhibits antiviral, antiproliferative and immunomodulation functions [3]. In vitro studies have shown that consensus interferon yields more potent antiviral and antiproliferative effects than other standard interferons (IFN-α2a, IFN-α2b, and pegylated versions) [6]. This artificial interferon was used in combination with ribavirin to treat nonresponder population or relapsers to a previous interferon regime [7,8]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
