基于MS2噬菌体lys基因的质粒型条件自溶菌的构建与应用

【背景】传统外源蛋白的原核表达通常需要以超声破碎或者酶解的方式破碎菌体，过程比较烦琐。【目的】构建基于MS2噬菌体lys基因的质粒型条件自溶菌，以简化外源蛋白的获取流程。【方法】从MS2噬菌体中克隆lys基因，构建重组表达质粒，并在大肠杆菌BL21(DE3)中异源表达，以此构建质粒型条件自溶菌，通过生长曲线和菌落形成单位反映自溶菌裂解效率，利用SDS-PAGE检测外源蛋白释放情况。【结果】构建了pBAD-lys BL21(DE3)、pBAD-Opti-lys BL21(DE3)及pCDF-BAD-Opti-lys BL21(DE3)这3种质粒型条件自溶菌。以上自溶菌在阿拉伯糖诱导后其宿主裂解效率均为99.99%以上，CFU结果显示含pCDF-BAD-Opti-lys质粒的宿主裂解效果更优，在此自溶菌BL21(DE3)中表达含His标签的重组绿色荧光蛋白(enhanced green fluorescent protein，eGFP)，经阿拉伯糖诱导后菌体中约63.00%以上的eGFP释放至胞外，利用Ni-NTA可以直接从培养基中纯化得到约30 kDa的单一目的蛋白。【结论】基于MS2噬菌体lys基因成功构建了阿拉伯糖诱导的质粒型条件自溶菌，此自溶菌能够以自我裂解的方式释放大部分胞内外源蛋白，简化传统外源蛋白获取流程。

Construction and application of plasmid-based opportunistic autolytic bacteria based on gene lys of MS2 bacteriophage

Background] Conventional prokaryotic expression of heterologous protein usually requires ultrasonication or enzymolysis to lyse bacteria, the process of which is quite cumbersome. Objective] We aimed to construct a type of plasmid-based opportunistic autolytic bacteria that exploited the lysis (lys) gene-mediated autolytic property of MS2 bacteriophage to facilitate the process of obtaining heterologous proteins. Methods] The gene lys was cloned from the MS2 bacteriophage into a recombinant expression plasmid, which was heterologously-expressed in Escherichia coli BL21(DE3), and a novel type of plasmid-based opportunistic autolytic bacteria was generated. By L-arabinose induction, the lytic efficiency of the above-mentioned bacteria was assessed by growth curves and colony forming units (CFUs). Finally, the release levels of the heterologous protein were examined by SDS-PAGE. Results] Three strains of plasmid-based opportunistic autolytic bacteria, pBAD- lys BL21(DE3), pBAD-Opti- lys BL21(DE3), and pCDF-BAD-Opti- lys BL21(DE3), were constructed. Following L-arabinose induction, all three strains of plasmid-based opportunistic autolytic bacteria achieved over 99.99% lytic efficiency. CFU data indicated that the host carrying pCDF-BAD-Opti- lys plasmid had better lytic effect than the other two. Upon L-arabinose-induced lysis of this host strain with heterologous expression of His-tagged enhanced green fluorescent protein (eGFP), more than 63% of eGFP were released to the outside of the cell and a single target protein of approximately 30 kDa was obtained directly from the culture medium by Ni-NTA. Conclusion] In this study, we have successfully constructed a type of lys -mediated, plasmid-based opportunistic autolytic bacteria, which is capable of releasing most of the cytosolic heterologous proteins through L-arabinose-induced autolysis, simplifying the traditional process to acquire heterologous proteins.