积磷小月菌调控蛋白Mlp21700的异源表达及其与多聚磷酸盐(Poly-P)代谢基因启动子的结合

【背景】积磷小月菌(Microlunatus phosphovorus)是重要的聚磷微生物,在好氧条件下积累聚磷酸盐并在厌氧条件分解聚磷酸盐,这个过程可能受到精细的基因调控。【目的】利用凝胶迁移实验(EMSA)分析调控蛋白Mlp21700结合的多聚磷酸盐(Poly-P)代谢基因启动子,找到Mlp21700可能的调控靶点。【方法】以积磷小月菌JN459菌株的基因组DNA为模板,PCR扩增Mlp21700编码序列,构建重组质粒p ET28a-21700并转化到大肠杆菌Transetta(DE3)菌株,诱导表达后采用非变性方法纯化获得Mlp21700融合蛋白。利用PCR方法分别扩增各个Poly-P代谢基因的启动子,用生物素标记后作为探针。采用EMSA分析Mlp21700在试验条件下是否结合启动子以及结合强度。【结果】DNA测序和酶切验证表明p ET28a-21700携带正确的Mlp21700编码序列。SDS-PAGE分析显示试验条件下Transetta(DE3)菌株大量表达可溶性Mlp21700,纯化的重组Mlp21700蛋白纯度大于90%,含量为0.64 mg/mL。EMSA分析表明在试验条件下Mlp21700能够结合Mlp26610基因ppgk和Mlp44770基因ppx的启动子。【结论】调控蛋白Mlp21700可能参与Mlp26610和Mlp44770基因的转录调控,进而调控Poly-P的分解过程。

Heterologous expression of Microlunatus phosphovorus regulatory protein Mlp21700 and its binding to promoters of Poly-P metabolic genes in vitro

Background] Microlunatus phosphovorus is one of the important phosphorus accumulating organisms. It can accumulate polyphosphate (Poly-P) aerobically and hydrolyze Poly-P anaerobically, a process fine-tuned by specific regulatory genes. Objective] To investigate Poly-P metabolic genes bound by Mlp21700, electrophoretic mobility shift assay (EMSA) was performed. Methods] The coding sequence of Mlp21700 was amplified and cloned into pET28a to generate a recombinant plasmid pET28a-21700 that was then transformed into Transetta(DE3) to express the recombinant protein Mlp21700. Mlp21700 was purified under non-denaturing conditions. Promoter sequences of Poly-P metabolic genes were amplified by PCR, labeled with biotin, and used as probes in EMSA assays to investigate binding of Mlp21700 to the above probes. Results] pET28a-21700 was confirmed by DNA sequencing and enzyme digestion. SDS-PAGE analysis indicated that Mlp21700 was highly expressed in soluble form. The purity of Mlp21700 surpassed 90% and the concentration reached 0.64 mg/mL. Our EMSA assays showed that Mlp21700 bound to the promoters of Mlp26610 (ppgk) and Mlp44770 (ppx). Conclusion] Mlp21700 may directly regulate Mlp26610 and Mlp44770, and thus regulate Poly-P metabolism.