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.
Zhou
Xuezhang
X
College of Life Science, Ningxia University, 539 W. Helanshan Road, Yinchuan, Ningxia 750021, China.
Song
Zhenwei
Z
Liu
Xiaoming
X
Jia
Fang
F
Wang
Yujiong
Y
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)