Production of recombinant porcine interferon alpha using PHB-intein-mediated protein purification strategy.
Interferons (IFNs) are involved in the pathogenesis and recovery of viral and other infectious diseases. Recombinant IFNs have been used as anti-infectious agents exhibiting a broad range of antiviral and immunomodulatory properties in both human and domestic animals. In this report, we describe a highly efficient and economical approach to purify porcine IFN alpha (PoIFNα) using polyhydroxybutyrate (PHB) as the affinity carrier and intein for self-cleaving removal of the affinity tag. Additionally, the conditions of protein expression and purification have been optimized. Our results suggested that culture medium containing 1.62% (w/v) of sodium lactate dramatically increases the accumulation of PHB binding protein in Escherichia coli cells. High yields of recombinant PoIFNα (30-35 mg/L, 97% purity by high-performance liquid chromatography) were obtained using intein-mediated self-cleaving conditions using a cleavage-inducing buffer with a pH of 6.5 at 20 °C for 24-36 h. The antiviral activity of the recovered recombinant PoIFNα was up to 1.4 x 10⁶ IU/mg of protein ascertained using recombinant human IFNα1 as a standard. This report also demonstrates that large-scale production of intein-mediated purification of highly pure and active recombinant PoIFNα is feasible for the purposes of experimental studies, veterinary clinic therapeutics, and swine infectious disease control.
College of Life Science, Ningxia University, 539 W. Helanshan Road, Yinchuan, Ningxia 750021, China.
Xuezhang
X
Zhou
Zhenwei
Z
Song
Xiaoming
X
Liu
Fang
F
Jia
Yujiong
Y
Wang
D000818:Animals; D002846:Chromatography, Affinity / Q000295:instrumentation / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D047668:Inteins; D016898:Interferon-alpha / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D011108:Polymers / Q000737:chemistry*; D000091402:Prohibitins; D015202:Protein Engineering; D019154:Protein Splicing; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013552:Swine
D016898:Interferon-alpha; C000717328:PHB protein, human; D011108:Polymers; D000091402:Prohibitins; D011993:Recombinant Fusion Proteins; C111948:poly(hydroxyethanoate)