title: Auto-induction expression of human consensus interferon-alpha in Escherichia coli.
abstract: BACKGROUND
  Isopropyl-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.
  
  
  RESULTS
  The 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.
  
  
  CONCLUSIONS
  The 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.
authors.affiliation: Therapeutic and Protective Proteins Laboratory, Protein Research Department, Genetic Engineering and Biotechnology Research Institute, City for Scientific Research and Technology Applications, New Borg EL-Arab, 21934, Alexandria, Egypt. mohamed_n8283@yahoo.com.
authors.forename: Nawal Abd
authors.initials: NA
authors.lastname: EL-Baky
authors.affiliation: College of Applied Medical Sciences, King Abdulaziz University, P.O. Box 80203, Jeddah, 21589, Saudi Arabia. DRMHL@HOTMAIL.COM.
authors.forename: Mustafa H
authors.initials: MH
authors.lastname: Linjawi
authors.affiliation: Therapeutic and Protective Proteins Laboratory, Protein Research Department, Genetic Engineering and Biotechnology Research Institute, City for Scientific Research and Technology Applications, New Borg EL-Arab, 21934, Alexandria, Egypt. redwan1961@yahoo.com.|Biological Sciences Department, Faculty of Science, King Abdulaziz University, P.O. Box 80203, Jeddah, 21589, Saudi Arabia. redwan1961@yahoo.com.
authors.forename: Elrashdy M
authors.initials: EM
authors.lastname: Redwan
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D000998:Antiviral Agents / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D002470:Cell Survival / Q000187:drug effects; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D056945:Hep G2 Cells; D018259:Herpesvirus 1, Human / Q000187:drug effects; D006801:Humans; D016898:Interferon-alpha / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism* / Q000494:pharmacology; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D015533:Transcriptional Activation; D014709:Vero Cells
chemical_list: D000970:Antineoplastic Agents; D000998:Antiviral Agents; D016898:Interferon-alpha; D011994:Recombinant Proteins; C050739:interferon alfacon-1