β变形菌多聚磷酸激酶PPK2的原核表达及纯化  

Prokaryotic Expression and Purification of Polyphosphate Kinase PPK2 from β Proteobacterium

作　　者：闫东科 宫艳超 吕平[1] 许凤霞 李冬 马素静 YAN Dong-ke;GONG Yan-chao;LV Ping;XU Feng-xia;LI Dong;MA Su-jing(College of Biological and Environmental Engineering/Tianjin Vocational Institute,Tianjin 300350,China;College of Environment and Chemical Engineering/Tianjin Bohai Vocational Technical College,Tianjin 300402,China)

机构地区：[1]天津职业大学生物与环境工程学院,天津300350 [2]天津渤海职业技术学院环境与化工学院,天津300402

出　　处：《山东农业大学学报（自然科学版）》2020年第2期212-216,共5页Journal of Shandong Agricultural University：Natural Science Edition

基　　金：天津职业大学科学研究基金(No.20181111)。

摘　　要：多聚磷酸激酶2(Polyphosphate kinase 2, PPK2)在众多病原菌的致病性中发挥重要作用,而人等后生动物体内未发现ppk2基因,因而PPK2是新型抗生素开发的靶点。为探索β变形菌CB中PPK2在大肠埃希菌中的可溶性表达,获得纯度、浓度均可用于蛋白质晶体学研究的PPK2蛋白。首先,采用生物信息学方法对PPK2蛋白的基本理化性质和二级结构进行预测,利用限制性核酸内切酶酶切位点NdeΙ和HindⅢ将ppk2基因插入到原核表达载体pET30a中,并通过全质粒酶切法和目的基因测序确认重组表达载体PPK2/pET30a的准确性;接下来,重组表达载体通过物理法转入大肠埃希菌BL21(DE3)菌株中,利用IPTG诱导目的蛋白PPK2在37℃、25℃和16℃下试表达,并通过SDS-PAGE电泳和Western blot鉴定分析表达产物;最后,利用3 L表达菌液进行放大培养,表达产物先后经Ni-IDA亲和色谱柱、阴离子交换色谱柱和凝胶过滤色谱柱分离纯化。结果显示:大肠原核表达系统可在16℃、终浓度为0.2m M·L-1的IPTG诱导下稳定高效表达以可溶性单体形式存在的,相对分子质量约为35 ku的PPK2蛋白;放大培养中产生的PPK2蛋白经系列色谱法纯化后浓度可达12 mg·mL-1,纯度为95%,可用于PPK2蛋白的结构和功能研究。Polyphosphate kinase PPK2 plays an important role in the pathogenicity of many pathogens, whereas, the absence of a ppk2 orthologue in humans makes it a potential drug target. This work aims to explore the soluble expression of the PPK2 of the β proteobacterium CB, and to achieve the purity and concentration of PPK2 can be used for Protein Crystallography. Initially, Characteristics and secondary structure of PPK2 protein were predicted bioinformatically, the ppk2 gene was inserted into the prokaryotic expression vector pET30 a through restriction endonuclease sites NdeΙ and HindⅢ, the accuracy of recombinant expression vector PPK2/pET30 a was confirmed via whole plasmid digestion and target gene sequencing analysis. Furthermore, the recombinant expression vector was physically transformed into E. coli BL21(DE3) to have a expression test, PPK2 was induced by IPTG under 37 ℃, 25 ℃, 16 ℃ respectively, then the target protein was identified by SDS-PAGE electrophoresis and Western blot analysis. Finally, after utilizing 3 L expression bacterial liquid to make scale-up culture, PPK2 protein was sequentially purified by Ni-IDA affinity column, anion exchange, and gel filtration chromatography. The expression of PPK2 protein was stable and efficient in the E. coli prokaryotic expression system and was confirmed in a soluble monomer state. The PPK2 protein of the β proteobacterium CB, which Mr was about 35 ku, was induced under final concentration 0.2 mM·L-1 IPTG and 16 ℃. After purification by series chromatography, the concentration of the purified PPK2 protein produced by scale-up culture was reached 12 mg·mL-1 with 95% purity, which can be used to study the structure and function of PPK2 protein.

关 键 词：β变形菌 多聚磷酸激酶PPK2 原核表达 纯化 

分 类 号：Q814.1[生物学—生物工程]