新型内含肽介导PHB系统表达纯化PoIFN-α的研究

为简化猪α干扰素蛋白（PoIFNα）的纯化步骤,以蛋白质自切割元件内含肽（Intein）替代蛋白酶,并以细菌自身产生的聚β-羟基丁酸酯（PHB）颗粒替代亲和配基,构建了新型内含肽介导PHB纯化PoIFNα的体系.结果显示,构建的基因工程大肠杆菌系统可成功实现PoIFNα的表达和纯化;φ（乳酸钠）=2.7%时,比较适宜工程菌的PHB累积;当诱导自切割反应温度为20℃,pH=6.5,反应时间为24~36 h时,可以实现内含肽C端高效自切割,纯化出的PoIFNα产量为20~30 mg/L.实验为重组猪α干扰素的工业化规模生产奠定了基础.

Research on Expression and Purification of Porcine Interferon Alpha in Escherichia coli with Novel Intein-mediated PHB System

To purify porcine interferon alpha（PoIFNα） in a very simple way,a recombinant gene engineer system was constructed.In this gene engineer E.coli,the self-splicing of intein can take place protease and the endogenous poly-β-hydroxy butyrate（PHB） granules which produced in the same cells can be as an affinity matrix to replace the traditional immobilized ligand.The results showed that PoIFNα can be expressed and purified by this constructed system,and the best concentration of sodium lactate for PHB granules production was 2.7%;when the temperature of solution was 20 ℃,pH was 6.5,and the self-splicing process lasted for 24-36 h,the intein can do well in releasing the target protein PoIFNα from its C-terminus for good yield at 20-30 mg/L.This work might be feasible for industrial-scale production of recombination protein,especially for the gene engineering drug porcine interferon alpha.