Zhiqiang Xiong. Tel:+86-21-55803272; E-mail:
研究精氨酸代谢调控蛋白ArgR对嗜热链球菌胞外多糖(EPS)合成的调控作用。
利用大肠杆菌异源表达嗜热链球菌ArgR蛋白,通过尿素变性-复性和Ni2+亲和层析纯化。采用凝胶电泳迁移(EMSA)和生物膜层干涉(BLI)分析ArgR和
大肠杆菌异源表达的ArgR为包涵体,使用尿素变性-复性纯化可获得2.95 mg/mL可溶性蛋白;EMSA和BLI结果显示ArgR和启动子PepsA有特异性结合,且结合因解离水平低而稳定;过表达
本研究表明ArgR能特异性结合嗜热链球菌
The regulatory effect of arginine regulator ArgR on the biosynthesis of exopolysaccharides (EPS) was studied in
ArgR from
Heterologous expression of ArgR was formed inclusion body, and 2.95 mg/mL of soluble protein was achieved by urea denaturation refolding. EMSA and BLI analysis showed that ArgR can specifically bind with the promoter PepsA and their affinity was high because of the low dissociation. Increased expression of
It is the first time to report that ArgR can specifically bind the promoter of
乳酸菌胞外多糖(exopolysaccharides, EPS)分为同聚多糖和杂聚多糖[
革兰氏阳性菌胞内精氨酸生物合成受
嗜热链球菌S-3是本实验室筛选的一株具有良好产粘性和口感辅助效果的益生乳酸菌。对S-3中EPS结构解析,发现其单糖组成为N-乙酰半乳糖胺、半乳糖和葡萄糖,摩尔比为1:2:1[
所用菌株与质粒如
本研究使用的菌株和质粒
Strains and plasmids in this study
Strains and plasmids | Characterizations | Sources |
Strains | ||
Top10 | Invitrogen | |
BL21 (DE3) | Invitrogen | |
S-3 | Our lab | |
Plasmids | ||
pET30α | Expressional vector, KanR, 5.4 kb | Novagen |
pIB184 | Shuttle vector, EmR, containing constitutive promoter P23 | BioVector |
pPH12 | pET30α containing N-6His- |
This study |
pPH13 | pET30α containing N, C-6His- |
This study |
pPH14 | pET30α containing C-6His- |
This study |
pPH15 | pIB184 containing |
This study |
pPH16 | pIB184 containing antisense |
This study |
质粒提取、DNA和PCR纯化回收试剂盒购自Axygen公司;高保真酶、限制性内切酶、T4连接酶和蛋白酶抑制剂购自宝生物工程(大连)有限公司和南京诺唯赞生物科技有限公司;Ni-NTA琼脂糖树脂购自QIAGEN公司;EMSA化学发光试剂盒购自上海碧云天生物技术有限公司;电泳仪设备及超灵敏凝胶成像仪购自Bio-Rad公司;分子互作仪及链霉亲和素传感器购自Fortebio公司。
引物由生工生物工程(上海)股份有限公司合成(
本研究使用的引物序列
Primers used in this study
Primer name | Sequence (5′→3′) |
Underlined parts are restriction sites, underlined parts are His-tag sequences, and underlined parts are homologous arms. | |
ArgR-1-F | GGAATTC |
ArgR-1-R | CCG |
ArgR-2-F | GGAATTC |
ArgR-2-R | CCG |
ArgR-3-F | GGAATTC |
ArgR-3-R | CCG |
box-F1 | |
box-R1 | |
box-R2 | ATATCAATCATTTAAATATTGTGAACTATCTTTTA |
box-F2 | TTAAATGATTGATATCATAATG |
box-R3 | |
Biotin-F-R | AGCCAGTGGCGATAAG |
ArgR-pIB-F | |
ArgR-pIB-R | |
antiArgR-pIB-R | |
antiArgR-pIB-R |
以S-3基因组为模板,使用引物ArgR-1-F/R、ArgR-2-F/R和ArgR-3-F/R扩增3种
将质粒pET30α、pPH12、pPH13和pPH14分别转化至
按照前述方法培养和诱导菌体,破碎离心后获得包涵体,使用包涵体溶解液(8 mol/L Urea,50 mmol/L NaH2PO4,300 mmol/L NaCl;pH 8.0)将其低温溶解,经0.45 μm滤膜过滤;用2个柱体积洗涤液(5 mmol/L Imidazole,50 mmol/L NaH2PO4,300 mmol/L NaCl;pH 8.0)平衡Ni-NTA琼脂糖树脂后,包涵体溶解液以0.5 mL/min的流速流过柱体;用5个柱体积平衡液(10 mmol/L Imidazole,8 mol/L Urea,50 mmol/L NaH2PO4,300 mmol/L NaCl;pH 8.0)以1 mL/min的流速洗去杂蛋白;用2个柱体积洗脱液(250 mmol/L Imidazole,8 mol/L Urea,50 mmol/L NaH2PO4,300 mmol/L NaCl;pH 8.0)洗脱出目的蛋白。使用3 kDa超滤管低温脱盐浓缩,测定蛋白质浓度,–80 ℃保存备用。
使用引物box-F1/R3以及Biotin-F-R扩增得到两端含有生物素标记的PepsA片段,同时获得未标记片段。凝胶阻滞实验(EMSA,electrophoresis mobility shift assays)如下:10 μL反应体系包含25 ng标记探针,不同浓度ArgR (0–10 μmol/L)和2 μL结合缓冲液,对照组需额外加入1.25 μg未标记探针;25 ℃孵育20 min后上样至4% (
生物素标记的PepsA片段使用DNA缓冲液(10 mmol/L HEPES,2 mmol/L MgCl2,0.1 mmol/L EDTA,200 mmol/L KCl;pH 8.0)溶解,ArgR溶液中加入终浓度1‰ (
PepsA启动子上预测含有2个ArgR结合位点(ARG-box),其中ARG-box1位于PepsA启动子上游38 bp,ARG-box1和ARG-box2间距79 bp。为得到PepsA上结合位点的突变DNA,以PepsA启动子为模板,使用引物box-F1/R1扩增得到ARG-box1缺失突变DNA;以引物box-F1/R2及box-F2/R3的扩增产物为模板,用引物box-F1/R3扩增得到ARG-box2缺失突变DNA;以引物box-F1/R2及box-F2/R1扩增产物为模板,用引物box-F1/R1扩增得到ARG-box1及ARG-box2缺失突变DNA。3种缺失突变DNA通过引物Biotin-F-R扩增标记生物素,采用EMSA检测ArgR与3种缺失突变DNA的结合情况。10 μL EMSA反应体系含25 ng标记探针和10 μmol/L ArgR,对照组不引入蛋白,竞争探针为1.25 μg。
重组菌株S-3/pPH15、S-3/pPH16和S-3/pIB184过夜培养的种子液分别以3%接种量接种于LM17培养基,培养至6、12、24 h离心收集上清液。根据已报道方法[
eps基因簇启动子PepsA生物素标记,结合链霉亲和素磁珠,通过Pull-down方法捕获到结合蛋白ArgR。在此基础上,为探究ArgR对嗜热链球菌EPS合成的转录调控作用,我们首先在大肠杆菌中异源表达ArgR。
质粒示意图以及ArgR表达和纯化
Plasmid structure and protein expression of ArgR. A: Structures of plasmid pPH12, pPH13 and pPH14. B: ArgR expression in
为增加可溶性蛋白表达,我们采用低温诱导降低ArgR蛋白合成速度,减少包涵体形成。在20 ℃使用不同IPTG浓度和诱导时间表达ArgR,分析条带灰度值的结果表明与其他两种IPTG浓度相比,在0.4 mmol/L IPTG浓度下ArgR可溶性表达量最高,在12 h时ArgR占比总蛋白达到82% (
不同诱导条件的ArgR蛋白表达及尿素变性纯化包涵体
ArgR expression and purification using urea. A: SDS-PAGE of ArgR expression at different IPTG concentrations and induction time. B: total soluble protein content under different induction conditions. C: purification of inclusion body protein of ArgR by urea. M: protein marker; lane 1: bacterial culture; lane 2, 3: supernatant and precipitation after fragmentation and centrifugation; lane 4: purified protein by urea.
为研究ArgR对
ArgR和PepsA启动子的相互作用
Interaction between ArgR and Promoter PepsA. A: The interaction by EMSA; Increasing amount of ArgR concentrations (0, 2, 4, 6, 8, 10 μmol/L) were used with the biotin-labeled probe at 25 ng; lane P: 1.25 μg unlabeled probe was added as the control. B: Kinetic curves measured by BLI with ArgR concentrations from 1 mg/mL to 0.0625 mg/mL, and all baselines leveled when balance and dissociation.
为进一步证明DNA结合位点对ArgR结合的影响,我们将PepsA中2个预测的ArgR结合位点ARG-box1和ARG-box2进行缺失突变后,与ArgR进行EMSA结合验证(
PepsA中ARG-box缺失突变对结合ArgR的影响
Effects of ARG-box mutation of PepsA on ArgR binding. Lane 1: original PepsA without ArgR; lane 2: original PepsA with ArgR; lane 3: original PepsA with ArgR and competitive DNA; lane 4: ARG-box1 deleted PepsA without ArgR; lane 5: ARG-box1 deleted PepsA with ArgR and competitive DNA; lane 6: ARG-box2 deleted PepsA without ArgR; lane 7: ARG-box2 deleted PepsA with ArgR and competitive DNA; lane 8: ARG-box1 and ARG-box2 deleted PepsA without ArgR; lane 9: ARG-box1 and ARG-box2 deleted PepsA with ArgR and competitive DNA.
为进一步证明
过表达和弱化
Effects on cell growth and EPS yield by overexpressed or weakened
本实验采用
[
嗜热链球菌CNRZ1066的ARG-box motif
The motif of ARG-box generated based on
Analysis of ArgR binding sites on
Strains | Site (in S-3) | Sequence (5′→3′) | Motif (5′→3′) | |
16471–16488 | 2.2×10–5 | TTATTATTATTATAACAT | WwTdWATAAWwATAvAdW | |
16568–16585 | 3.9×10–5 | TTTATATCATTTTCCATT | ||
16568–16585 | 9.8×10–5 | TTTATATCATTTTCCATT | WnTGnATWWWWATnCAnW | |
16568–16585 | 4.8×10–5 | TTTATATCATTTTCCATT | nkTGyATAnTtTTnCnnG | |
16600–16617 | 4.8×10–5 | CTTTTACATTTTTAGTAG | ||
16471–16488 | 1.4×10–4 | TTATTATTATTATAACAT | AwwGwATAAWWATrCWnw | |
16568–16585 | 5.0×10–4 | TTTATATCATTTTCCATT |
ArgR蛋白作为转录调控因子在不同代谢途径中调控作用不同。例如,在猪链球菌中ArgR蛋白作为转录激活因子,激活精氨酸合成代谢中
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