Name: expression, purification, and antimicrobial activity of s100a12
Uploaded: 2017-05-13T13:00:00
Description: Watch this Scientific Journal Video about Expression, Purification, and Antimicrobial Activity of S100A12 at JoVE.com

这项研究得到了退伍军人事务部职业发展奖1IK2BX001701，国家推进转化科学中心的CTSA奖UL1TR000445，国家科学基金会奖1547757和1400969以及NIH授予GM05551的支持。其内容完全是作者的责任，不一定代表国家推进转化科学中心或国家卫生研究院的官方观点。

S100A12的表达<ol><li>使用标准热休克方案，用pGEMEX-S100a12质粒转化感受态BL21 DE3细胞。在微量离心管中在冰上向50μL细菌中加入1至5μL质粒。孵育20分钟</li><li>在42℃下将细胞热冲击30秒。 </li><li>将细胞在冰上孵育2分钟。 </li><li>向细胞中加入500μLSOC介质。在37℃下，在轨道振荡器上以250rpm振荡孵育1小时。 </li><li>在LB-琼脂培养基（补充100μg/ mL氨苄青霉素）上150μL的转化反应。在37°C孵育12-16 h。 </li><li...

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提出了一种用于人S100A12的表达和纯化的有效方案。 大肠杆菌表达系统是用于生产重组蛋白的最常用工具，特别是生物化学和生物物理学研究需要mg量时。这里描述的程序的重要增强是使用自动诱导培养基26 ，其与标准的Luria-broth培养基18的表达相比将纯化蛋白的产量提高了近三十倍。另外，自动感应介质大大简化了蛋白质表达工作流程。使用常规生长培?...

S100蛋白是一类具有多种功能的钙结合蛋白的EF手系家族1 。它们以组织和细胞特异性方式表达，并调节广泛的细胞功能2,3 。钙结合蛋白独特，S100蛋白表现出细胞内和细胞外功能4,5 。在细胞内，Ca 2+结合诱导构象变化，其暴露出特异性靶向蛋白质结合配偶体的疏水性表面。这种细胞内机制调节重要的过程，如细胞增殖，分化和能量代谢。在细胞外环境中，S100蛋白表现出两个功能7 。一方面，它们作为损伤相关分子模式（DAMP）蛋白质，并引发促炎症通过与模式识别受体8,9的相互作用产生免疫反应。此外，S100蛋白质类螯合物的几个成员过渡金属，这是一种功能，用于在称为营养免疫的过程中将微生物病原体饥饿10,11 。 S100A12（也称为钙粒蛋白C和EN-RAGE）在巨噬细胞和嗜中性粒细胞中高度表达，并且已被鉴定为炎性疾病的潜在生物标志物12,13 。除了在其EF手部位点处结合钙之外，S100A12还具有位于二聚体界面14,15相对端的两个高亲和力的过渡金属结合位点。每个结合位点由三个组氨酸残基和一个天冬氨酸残基组成，可螯合锌或铜sup class =“xref”&gt; 16,17。最近，我们报道S100A12依赖性锌饥饿在调节幽门螺杆菌生长和促炎毒力因子的活性中是重要的18 。 幽门螺杆菌感染世界人口约一半的胃;使其成为最成功的细菌病原体之一19 。 幽门螺杆菌感染可导致胃炎，胃肠炎，十二指肠溃疡，粘膜相关淋巴组织（MALT）淋巴瘤，侵袭性胃腺癌（胃癌）等胃肠疾病。胃癌是世界上非贲门癌相关死亡的主要原因，胃癌的单一最大相关危险因素是幽门螺杆菌感染 。 幽门螺杆菌仍然存在于胃的利基，尽管如此对病原体的免疫应答，强调需要更好地了解控制这种细菌感染的免疫机制20,21,22,23。 幽门螺杆菌相关炎症的特征在于多形核细胞或嗜中性粒细胞的深度浸润，其在感染部位18,24,25上沉积抗微生物蛋白质（包括S100A12）的所有组成成分。为了了解宿主和病原体之间的复杂对话，我们力求改进纯化S100A12的技术，并用它来研究其对该医学相关病原体的抗菌作用。下面的方案概述了其生物活性状态下S100A12纯化的改进技术;能够与高亲和力结合营养金属并将它们从侵入的微生物中螯合。此外，以下方法突出了该蛋白质作为关键试剂的用途，以研究天然抗微生物分子限制细菌病原体生长的机制。 S100A家族蛋白作为参与免疫信号传导和宿主防御的重要的先天免疫系统分子群已经获得了欣赏27 。其中最为深入的研究是钙卫蛋白（MRP-8/14，钙粒蛋白A / B，S100A8 / A9） 28,29,30 。钙卫蛋白是中性粒细胞相关蛋白，其形成S100A8和S100A9亚基的异二聚体，其在二聚体界面31处结合过渡金属。钙卫蛋白已显示具有两个金属结合位点：位点1可以结合Zn 2+ ，Mn 2+或Fe 2+ ，而Site 2可以<html>结合Zn 2+ 31,32 。许多报告已经证明，钙卫蛋白具有抗多种病原体的抗菌活性 ， 包括金黄色葡萄球菌 ， 白色念珠菌 ， 鲍曼不 动杆菌 ， 肺炎克雷伯杆菌 ， 大肠杆菌和幽门螺杆菌 ，并且抑制作用是由于钙卫蛋白25,28的金属螯合活性， 29 。 以前的工作证明钙卫蛋白在幽门螺杆菌上发挥了许多活性，包括改变外膜中的脂质A结构，抑制cag- IV型分泌系统（其是幽门螺杆菌中的主要促炎毒力因子），诱导生物膜形成和抑制幽门螺杆菌生长和活力以剂量依赖的方式class =“xref”&gt; 25，33。此外，遗传和生物化学测定显示钙卫蛋白对幽门螺杆菌的抗菌活性主要来源于其与营养锌25的结合能力。 幽门螺杆菌需要锌，如先前的研究所确定的，其利用化学成分确定的培养基来确定该病原体生长和增殖的微量营养素需求34 。此外，钙卫蛋白在幽门螺杆菌感染的组织中高度丰富，并与嗜中性粒细胞浸润相关，表明宿主可能在感染和随后的炎症中使用钙卫蛋白作为抗微生物策略。 来自无偏蛋白质组学筛选技术的最新证据表明，在活性氧丰富的条件下，calprotec锡经历改变六组氨酸结合位点的翻译后修饰，从而抑制蛋白36的金属结合活性。因此，我们假设在这些分子的广泛谱系中的其他S100A家族蛋白可能潜在地作为辅助金属螯合剂。我们选择S100A12进行进一步研究，因为在上述翻译后修饰屏幕中没有鉴定，它具有结合锌的能力，并且在来自幽门螺旋杆菌感染个体的人体组织中高度丰富。 我们的工作表明，S100A12可以在幽门螺杆菌 G27菌株中以剂量依赖的方式抑制幽门螺杆菌生长和活力，并且通过添加过量的营养锌可以逆转该蛋白的抗微生物活性。这项工作补充了我们以前的工作，表明S100A12对PMSS1和7.1产生抗菌活性3株幽门螺杆菌 ，证明其对许多临床分离株和实验室适应的幽门螺杆菌菌株18的广谱抗菌活性。一起，这些结果证实了S100A12作为通过营养免疫来控制细菌生长和增殖的机制的重要性。未来对这种重要的宿主 - 细菌相互作用的研究可能包括利用S100A12的活性来减少宿主组织内的细菌负担，或者确定这种蛋白质在幽门螺旋杆菌感染背景下对免疫信号传导的贡献。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>S100A12 Expression</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Expression cells</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BL 21 (DE3) competent cells</td>
			<td>New England Biolabs</td>
			<td>C25271</td>
		</tr>
		<tr>
			<td>Autoinduction medium components</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast Extract</td>
			<td>Research Products International</td>
			<td>Y20020-250.0</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Research Products International</td>
			<td>S23020-500.0</td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>Research Products International</td>
			<td>T60060-250.0</td>
		</tr>
		<tr>
			<td>FeCl3</td>
			<td>Sigma-Aldrich</td>
			<td>F2877</td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>Research Products International</td>
			<td>M65240-100.0</td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>Research Products International</td>
			<td>S23100-500.0</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Research Products International</td>
			<td>P41200-500.0</td>
		</tr>
		<tr>
			<td>NH4Cl</td>
			<td>Research Products International</td>
			<td>A20424-500.0</td>
		</tr>
		<tr>
			<td>Na2SO4</td>
			<td>Research Products International</td>
			<td>S25150-500.0</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Research Products International</td>
			<td>G22020-1.0</td>
		</tr>
		<tr>
			<td>D-glucose</td>
			<td>Research Products International</td>
			<td>G32040-500.0</td>
		</tr>
		<tr>
			<td>lactose</td>
			<td>Sigma-Aldrich</td>
			<td>L2643</td>
		</tr>
		<tr>
			<td>Selection agent</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ampicillin</td>
			<td>Research Products International</td>
			<td>A40040-5.0</td>
		</tr>
		<tr>
			<td>S100A12 Purification</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AKTA Start Chromatography System</td>
			<td>GE</td>
			<td>29-220-94</td>
		</tr>
		<tr>
			<td>HiTrap Q Sepharaose Fast Flow</td>
			<td>GE</td>
			<td>17-5156-01</td>
		</tr>
		<tr>
			<td>HiPrep 16/60 S-200 HR</td>
			<td>GE</td>
			<td>17-1166-01</td>
		</tr>
		<tr>
			<td>Nanodrop lite spectrophotometer</td>
			<td>Thermo Fisher Scientific</td>
			<td>ND-LITE</td>
		</tr>
		<tr>
			<td>Tris Base</td>
			<td>Research Products International</td>
			<td>T60040-1000.0</td>
		</tr>
		<tr>
			<td>H. pylori Culture</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Blood agar plates</td>
			<td>Lab Supply Company</td>
			<td>BBL221261</td>
		</tr>
		<tr>
			<td>Brucella broth</td>
			<td>Sigma-Aldrich</td>
			<td>B3051</td>
		</tr>
		<tr>
			<td>Cholesterol (250X)</td>
			<td>Thermo Fisher Scientific</td>
			<td>12531018</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>793566</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>C4901</td>
		</tr>
		<tr>
			<td>&#946;-mercaptoethanol</td>
			<td>Sigma-Aldrich</td>
			<td>M6250</td>
		</tr>
		<tr>
			<td>Tris Base</td>
			<td>Sigma-Aldrich</td>
			<td>T1503</td>
		</tr>
		<tr>
			<td>Zinc Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>229997</td>
		</tr>
	</tbody>
</table>

expression, purification, and antimicrobial activity of s100a12

钙粒蛋白蛋白是先天免疫的重要介质，是钙结合蛋白的EF手家族的S100类的成员。一些S100蛋白质具有以高亲和力结合过渡金属的能力，并且在称为“营养免疫”的过程中有效地将其远离入侵的微生物病原体。 S100A12（EN-RAGE）结合锌和铜，并且在先天免疫细胞如巨噬细胞和嗜中性粒细胞中是高度丰富的。我们报告了一种用于表达，富集和纯化其活性金属结合构型的S100A12的精制方法。这种蛋白质在细菌生长和活力分析中的应用揭示了S100A12对细菌病原体幽门螺杆菌具有抗菌活性。抗菌活性取决于S100A12的锌结合活性，S100A12螯合营养锌，从而使需要锌生长的幽门螺杆菌饥饿roliferation。

S100A12表达纯化三步纯化从50mL细菌培养物中产生约40mg的重组S100A12。第一步是内源性大肠杆菌蛋白质的硫酸铵沉淀。该步骤之后是阴离子交换层析（ 图 1A ）。通过用考马斯亮蓝染色的SDS-PAGE追踪蛋白质（ 图1B ）。纯化方法的最后一步是将含有S100A12的合并级分用于大小排阻色谱法，用?...

Watch this Scientific Journal Video about Expression, Purification, and Antimicrobial Activity of S100A12 at JoVE.com

Expression, Purification, and Antimicrobial Activity of S100A12

在这里，我们提出了一种表达和纯化S100A12（钙粒蛋白C）的方法。我们描述了一种测定其对人类幽门螺杆菌的抗微生物活性的方案 。

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