酿酒酵母表达和纯化功能性的铁载体合成蛋白PchE

Heterologous expression and purification of the activated siderophore synthetase PchE in Saccharomyces cerevisiae

【目的】利用酿酒酵母表达系统,通过乙醇脱氢酶启动子异源表达细菌源的铁载体合成蛋白PchE,并与来源于枯草芽孢杆菌的泛酰化酶Sfp同宿主共表达,探索真核表达体系表达具有生化活性的细菌源蛋白。【方法】从大肠杆菌BAP 1染色体上扩增sfp基因,将pchE基因及串联的pchE与sfp基因分别构建到酵母-大肠杆菌穿梭质粒pXW55中,各自转化酿酒酵母BJ5464-npgA表达,经过亲和层析和离子交换层析纯化蛋白,利用HPLC检测细菌源与酵母源表达的PchE在体外重构生化反应中的催化活性。【结果】利用酿酒酵母表达系统可以获得高纯度的原核蛋白PchE。真菌源的泛酰化基因NpgA和细菌源的Sfp,均可泛酰化修饰PchE,合成中间产物HPT-Cys。【结论】在酿酒酵母Saccharomyces cerevisiae BJ5464-npgA表达系统中,首次证明真菌源的泛酰化基因NpgA和细菌源的Sfp,均可泛酰化修饰细菌源的非核糖体肽合酶。比较酵母和细菌宿主的目标蛋白表达,证明酵母表达的巨大蛋白PchE的纯度更高,非特异性条带减少,推测酵母宿主可能更适合表达纯化功能性的巨型蛋白质。

[Objective] Expressing the bacterial siderophore synthetase PchE under the ADH2 promoter in Saccharomyces cerevisiae system with the PPTase Sfp from Bacillus subtilis, to explore the heterologous expression of activated bacterial protein in eukaryotic system. [Methods] The sfp gene was amplified from Escherichia coli BAP 1. Both pchE gene and pchE tandem with sfp were cloned to the yeast-E. coli shuttle vector pXW55, and transformed into the Saccharomyces cerevisiae BJ5464-npgA strain. After the purification steps including affinity chromatography and ion-exchange chromatography, HPLC was used to detect whether the PchE from E. coli and Saccharomyces cerevisiae maintain catalytic activity in the biochemical reaction in vitro. [Results]In the Saccharomyces cerevisiae expression system, prokaryotic protein PchE was obtained with high purity. And no matter assisted by the bacterial or fungal PPTase, PchE can be modified and synthesize intermediate product HPT-Cys. [Conclusion] It was firstly demonstrated that both fungal gene npgA and bacterial gene sfp can modify bacterial nonribosomal peptide synthase. With the comparison of protein expression between yeast and bacterial host, the giant protein PchE expressed by yeast has higher purity and fewer nonspecific bands. This suggested that yeast host might be more suitable for expressing and purifying functional giant protein.