He Jia-Hui, Sun Jie-Li, Yan Wen-Juan, Wang Fang
Department of Stomatology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China.E-mail:
Nan Fang Yi Ke Da Xue Xue Bao. 2017 May 20;37(5):633-639. doi: 10.3969/j.issn.1673-4254.2017.05.11.
To identify the functions of the proteins containing the GGDEF or EAL domain in Lactobacillus acidophilus for investigation of the regulatory mechanism of c-di-GMP in this strain.
The DNA fragments of NH13_07045-GGDEF, NH13_07050 and NH13_07055 from Lactobacillus acidophilus ATCC4356 were amplified by PCR and cloned into the expression vector pMAL-His-c2. After sequencing, the recombinant plasmids were transformed into competent Escherichia coli cells, which were induced by IPTG to express the recombinant proteins fused with maltose binding protein (MBP). The fusion proteins were purified using amylose resin column for diguanylate cyclase (DGC) or phosphodiesterase (PDE) activity assays in vitro followed by analysis with high-performance liquid chromatography (HPLC).
The target DNA fragments were obtained by PCR, and their sequences were all identical to that in GenBank. The purified and concentrated fusion proteins, which were identified by SDS-PAGE and Western blotting, had relative molecular masses of 59 kD, 67 kD and 72 kD. HPLC analysis showed no DGC activity in NH13_07045-GGDEF, while PDE activity was found in NH13_07050 but not in NH13_07055.
We obtained the protein encoded by NH13_07050 that possesses PDE activity in vitro. This protein may facilitate the evaluation of the regulatory function of c-di-GMP in Lactobacillus acidophilus.
鉴定嗜酸乳杆菌中含GGDEF或EAL结构域的蛋白质的功能，以研究该菌株中环状二鸟苷单磷酸（c-di-GMP）的调控机制。
通过聚合酶链反应（PCR）扩增嗜酸乳杆菌ATCC4356中NH13_07045-GGDEF、NH13_07050和NH13_07055的DNA片段，并克隆到表达载体pMAL-His-c2中。测序后，将重组质粒转化到感受态大肠杆菌细胞中，用异丙基-β-D-硫代半乳糖苷（IPTG）诱导表达与麦芽糖结合蛋白（MBP）融合的重组蛋白。使用直链淀粉树脂柱纯化融合蛋白，用于体外双鸟苷酸环化酶（DGC）或磷酸二酯酶（PDE）活性测定，随后用高效液相色谱（HPLC）进行分析。
通过PCR获得了目标DNA片段，其序列与GenBank中的序列完全相同。经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和蛋白质免疫印迹法（Western blotting）鉴定，纯化和浓缩后的融合蛋白相对分子质量分别为59 kD、67 kD和72 kD。HPLC分析显示，NH13_07045-GGDEF无DGC活性，而NH13_07050有PDE活性，NH13_07055无PDE活性。
我们获得了体外具有PDE活性的由NH13_07050编码的蛋白质。该蛋白可能有助于评估嗜酸乳杆菌中c-di-GMP的调控功能。
