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>挑一个殖民地接种2 mL LB（补充100μg/ mL氨苄青霉素）。 </li><li>在37℃下在轨道振荡器（300rpm）上孵育4-6小时。 OD 600应读数在1-3个吸光度单位之间。 </li><li>加入500μL起始培养物至50 mL ZYM-5052 autoindu补充有100μg/ mL氨苄青霉素的补充培养基26 。为了获得最佳曝气和最大表达，请使用250 mL带挡板的锥形瓶。在37℃下摇动（300rpm）24小时。 </li><li>将细菌悬浮液转移到离心管中。在4℃下通过离心（4,000 xg，10分钟）将细胞造粒。 </li><li>将介质倾倒，记录样品，并将细胞粘贴存储在-80°C。样品多年稳定。 </li></ol> 2.使用低压色谱法纯化S100A12 <ol><li>将细胞重悬于30 mL 20 mM Tris，pH 8.0中。 </li><li>在冰上超声处理悬浮液以溶解细胞。使用〜20 W输出，5 s on和5 s off循环5 min。 </li><li>将溶液转移到高速离心管中。通过离心澄清细胞裂解物，在4℃下20,000×g 30分钟。 </li><li>倾析上清液并转移到干净的100mL聚丙烯烧杯中。通过将烧杯放在冰上来冷却溶液。添加一个搅拌缸并慢慢加入11.20g硫酸铵。让溶液在冰上再搅拌1小时。这将产生60％的硫酸铵溶液并沉淀大部分大肠杆菌内源性蛋白质。 S100A12将保持溶解。 </li><li>在4℃，20,000×g离心20分钟以沉淀沉淀的蛋白质。 </li><li>倾倒上清液并转移到透析管（MWCO 3,500 kDa）。在4℃下对1μL的20mM Tris，pH 8.0进行透析。更换透析缓冲液两次。允许4小时之间的变化。 </li><li> 阴离子交换层析 <ol><li>在低压系统上进行色谱。典型流速为1 mL / min。 </li><li>用10mL 20mM Tris，pH 8.0平衡5mL Sepharose柱。 </li><li>使用样品泵装载〜40 mL的S100A12溶液（收集流量）。 </li><li>用10mL 20mM Tris，pH8洗涤柱。 </li><li>用0-30％梯度（Bub B为20mM Tris，pH 8.0,1M NaCl），经19柱体积（CV，95mL）。收集5 mL级分。 </li><li>取每个级分的10μL等分试样，并用考马斯染色的MES SDS PAGE进行分析。使用恒定电压（20 V / cm）运行凝胶30分钟。 </li><li>含有S100A12的游泳池部分。 S100A12在变性凝胶上以约10kDa的蛋白质运行。 </li><li>使用超滤装置（MWCO 10kDa）浓缩至5mL。离心机在3,000 xg〜8分钟。取最高分数。 </li></ol></li><li> 大小排阻层析 <ol><li>用1 CV（120 mL）的20mM Tris pH8,100mM NaCl平衡S75柱。 </li><li>注入&lt;5 mL浓缩的S100A12级分（来自离子交换层析）。 </li><li>以120mL的流速以1mL / m的流速开发柱。收集5 mL级分。 </li><li>取每个级分的10μL等分试样，并用考马斯染色的SDS PAGE进行分析。使用蛋白质鉴定蛋白质印迹或质谱（计算的单体亚基的分子量为10,575.0Da，测量值为10575.4Da）。 </li></ol></li><li> 池分数 <ol><li>使用分光光度计测量蛋白A 280的吸光度。使用SEC缓冲区作为空白。计算出的S100A12同型二聚体的消光系数为5960M - 1cm -1 。 注意:每50 mL培养物的典型产量为35-45 mg的S100A12。 </li><li>将等分试样S100A12放入1.5 mL微量离心管（1 mg /管）中，在液氮中快速冷冻，储存于-80°C。 </li></ol></li></ol> 3.抗菌活性测定<ol><li>将条纹幽门螺杆菌菌株G27置于补充有5％绵羊血液（血琼脂平板）的胰蛋白酶大豆琼脂平板上。在37℃下在补充有5％二氧化碳的室内空气中生长2-3天。 </li><li>将幽门螺杆菌接种到补充有1x胆固醇的布鲁杆菌肉汤中。文化过夜间在37℃下，在补充有5％二氧化碳的室内空气中摇动（250rpm）。 </li><li>将幽门螺杆菌 1:10稀释成50％布鲁氏菌肉汤，50％钙卫蛋白缓冲液加1x胆固醇，培养液中单独培养，或补充100μM氯化锌加0,100或1000μg/ mL纯化的S100A12。培养过夜振荡（250rpm），37℃，室温下补充5％二氧化碳。 </li><li>第二天，连续稀释并平板至血琼脂平板上。允许细菌菌落在37℃下在补充有5％二氧化碳的室内空气中生长2-3天。在存在或不存在S100A12和/或外源性锌的情况下，列举菌落形成单位以计算细菌生长。 </li></ol>

<ol>
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作者宣称他们没有竞争的经济利益。

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 。

S100A12的表达，纯化和抗菌活性

Calgranulin proteins are important mediators of innate immunity and are members of the S100 class of the EF-hand family of calcium binding proteins. Some ...

expression-purification-and-antimicrobial-activity-of-s100a12

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8.3K 次观看.  Fisk University. 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 。 

视频: Expression, Purification, and Antimicrobial Activity of S100A12

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

The serotonin transporter is a sodium and chloride-coupled transporter that &#34;pumps&#34; extracellular serotonin into cells. S-citalopram is a drug used to treat depression and anxiety by binding to the serotonin transporter with high-affinity, blocking serotonin reuptake. Here we report an efficient procedure and a set of tools to stabilize, express, purify, and crystallize serotonin transporter-antibody complexes bound to S-citalopram and other antidepressants. Mutations which stabilize the serotonin transporter were identified using an S-citalopram binding assay. Serotonin transporter expressed in baculovirus-transduced HEK293S GnTI- cells, was reconstituted into proteoliposomes and used to raise high-affinity antibodies. We have developed a strategy to discover antibodies that are useful for structural studies. A straightforward approach for the expression of antibody fragments in Sf9 cells has also been established. Transporter-antibody complexes purified using this procedure are well-behaved and readily crystallize, producing complexes with S-citalopram that diffract X-rays to 3-4 &#197; resolution. The strategies developed here can be utilized to determine the structure of other challenging membrane proteins.

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

This manuscript describes how to screen for thermostabilizing mutations, purify the human serotonin transporter, generate high affinity antibodies, and crystallize the serotonin transporter-antibody complex bound to the antidepressant drug S-citalopram. This protocol can be adapted to the study of other challenging membrane transporters, receptors, and channels.

热稳定，表达，纯化，和人类血清素转运蛋白结晶绑定到<em&gt;取值</em&gt; -citalopram

The serotonin transporter is a sodium and chloride-coupled transporter that &#34;pumps&#34; extracellular serotonin into cells. S-citalopram is a drug ...

科研

生物化学

Laboratory Scale Production and Purification of a Therapeutic Antibody

Ensuring the successful production of a therapeutic antibody begins early on in the development process. The first stage is vector expression of the antibody genes followed by stable transfection into a suitable cell line. The stable clones are subjected to screening in order to select those clones with desired production and growth characteristics. This is a critical albeit time-consuming step in the process. This protocol considers vector selection and sourcing of antibody sequences for the expression of a therapeutic antibody. The methods describe preparation of vector DNA for stable transfection of a suspension variant of human embryonic kidney 293 (HEK-293) cell line, using polyethylenimine (PEI). The cells are transfected as adherent cells in serum-containing media to maximize transfection efficiency, and afterwards adapted to serum-free conditions. Large scale production, setup as batch overgrow cultures is used to yield antibody protein that is purified by affinity chromatography using an automated fast protein liquid chromatography (FPLC) instrument. The antibody yields produced by this method can provide sufficient protein to begin initial characterization of the antibody. This may include in vitro assay development or physicochemical characterization to aid in the time-consuming task of clonal screening for lead candidates. This method can be transferable to the development of an expression system for the production of biosimilar antibodies.

This protocol describes the production of a therapeutic antibody in a mammalian expression system. The methods described include preparation of vector DNA, stable transfection and serum-free adaptation of a human embryonic kidney 293 cell line, set up of large scale cultures and purification using affinity chromatography.

实验室规模的生产和治疗性抗体的纯化

Ensuring the successful production of a therapeutic antibody begins early on in the development process. The first stage is vector expression of the ...

Purification and Reconstitution of TRPV1 for Spectroscopic Analysis

Polymodal ion channels transduce multiple stimuli of different natures into allosteric changes; these dynamic conformations are challenging to determine and remain largely unknown. With recent advances in single-particle cryo-electron microscopy (cryo-EM) shedding light on the structural features of agonist binding sites and the activation mechanism of several ion channels, the stage is set for an in-depth dynamic analysis of their gating mechanisms using spectroscopic approaches. Spectroscopic techniques such as electron paramagnetic resonance (EPR) and double electron-electron resonance (DEER) have been mainly restricted to the study of prokaryotic ion channels that can be purified in large quantities. The requirement for large amounts of functional and stable membrane proteins has hampered the study of mammalian ion channels using these approaches. EPR and DEER offer many advantages, including determination of the structure and dynamic changes of mobile protein regions, albeit at low resolution, that might be difficult to obtain by X-ray crystallography or cryo-EM, and monitoring reversible gating transition (i.e., closed, open, sensitized, and desensitized). Here, we provide protocols for obtaining milligrams of functional detergent-solubilized transient receptor potential cation channel subfamily V member 1 (TRPV1) that can be labeled for EPR and DEER spectroscopy.

This article describes specific methods to obtain biochemical quantities of detergent-solubilized TRPV1 for spectroscopic analysis. The combined protocols provide biochemical and biophysical tools that can be adapted to facilitate structural and functional studies for mammalian ion channels in a membrane-controlled environment.

光谱分析 TRPV1 的纯化与重构

Polymodal ion channels transduce multiple stimuli of different natures into allosteric changes; these dynamic conformations are challenging to determine ...

Expression and Purification of Mammalian Bestrophin Ion Channels

The human genome encodes four bestrophin paralogs, namely BEST1, BEST2, BEST3, and BEST4. BEST1, encoded by the BEST1 gene, is a Ca2+-activated Cl- channel (CaCC) predominantly expressed in retinal pigment epithelium (RPE). The physiological and pathological significance of BEST1 is highlighted by the fact that over 200 distinct mutations in the BEST1 gene have been genetically linked to a spectrum of at least five retinal degenerative disorders, such as Best vitelliform macular dystrophy (Best disease). Therefore, understanding the biophysics of bestrophin channels at the single-molecule level holds tremendous significance. However, obtaining purified mammalian ion channels is often a challenging task. Here, we report a protocol for the expression of mammalian bestrophin proteins with the BacMam baculovirus gene transfer system and their purification by affinity and size-exclusion chromatography. The purified proteins have the potential to be utilized in subsequent functional and structural analyses, such as electrophysiological recording in lipid bilayers and crystallography. Importantly, this pipeline can be adapted to study the functions and structures of other ion channels.

The purification of ion channels is often challenging, but once achieved, it can potentially allow in vitro investigations of the functions and structures of the channels. Here, we describe the stepwise procedures for the expression and purification of mammalian bestrophin proteins, a family of Ca2+-activated Cl- channels.

哺乳动物 Bestrophin 离子通道的表达与纯化

The human genome encodes four bestrophin paralogs, namely BEST1, BEST2, BEST3, and BEST4. BEST1, encoded by the BEST1 gene, is a Ca2+-activated Cl- ...

Caffeine Extraction, Enzymatic Activity and Gene Expression of Caffeine Synthase from Plant Cell Suspensions

Caffeine (1,3,7-trimethylxanthine) is a purine alkaloid present in popular drinks such as coffee and tea. This secondary metabolite is regarded as a chemical defense because it has antimicrobial activity and is considered a natural insecticide. Caffeine can also produce negative allelopathic effects that prevent the growth of surrounding plants. In addition, people around the world consume caffeine for its analgesic and stimulatory effects. Due to interest in the technological applications of caffeine, research on the biosynthetic pathway of this compound has grown. These studies have primarily focused on understanding the biochemical and molecular mechanisms that regulate the biosynthesis of caffeine. In vitro tissue culture has become a useful system for studying this biosynthetic pathway. This article will describe a step-by-step protocol for the quantification of caffeine and for measuring the transcript levels of the gene (CCS1) encoding caffeine synthase (CS) in cell suspensions of C. arabica L. as well as its activity.

This protocol describes an efficient methodology for the extraction and quantification of caffeine in cell suspensions of C. arabica L. and an experimental process for evaluating the enzymatic activity of caffeine synthase with the expression level of the gene that encodes this enzyme.

植物细胞悬浮液中咖啡因的提取、酶活性及咖啡因合成酶基因的表达

Caffeine (1,3,7-trimethylxanthine) is a purine alkaloid present in popular drinks such as coffee and tea. This secondary metabolite is regarded as a ...

Anaerobic Protein Purification and Kinetic Analysis via Oxygen Electrode for Studying DesB Dioxygenase Activity and Inhibition

Oxygen-sensitive proteins, including those enzymes which utilize oxygen as a substrate, can have reduced stability when purified using traditional aerobic purification methods. This manuscript illustrates the technical details involved in the anaerobic purification process, including the preparation of buffers and reagents, the methods for column chromatography in a glove box, and the desalting of the protein prior to kinetics. Also described are the methods for preparing and using an oxygen electrode to perform kinetic characterization of an oxygen-utilizing enzyme. These methods are illustrated using the dioxygenase enzyme DesB, a gallate dioxygenase from the bacterium Sphingobium sp. strain SYK-6.

Here we present a protocol for anaerobic protein purification, anaerobic protein concentration, and subsequent kinetic characterization using an oxygen electrode system. The method is illustrated using the enzyme DesB, a dioxygenase enzyme which is more stable and active when purified and stored in an anaerobic environment.

氧电极对 DesB 酶活性和抑制作用的厌氧蛋白纯化及动力学分析

Oxygen-sensitive proteins, including those enzymes which utilize oxygen as a substrate, can have reduced stability when purified using traditional aerobic ...

Expression and Purification of the Human Lipid-sensitive Cation Channel TRPC3 for Structural Determination by Single-particle Cryo-electron Microscopy

Transient receptor potential channels (TRPCs) of the canonical TRP subfamily are nonselective cation channels that play an essential role in calcium homeostasis, particularly store-operated calcium entry, which is critical to maintaining proper function of synaptic vesicle release and intracellular signaling pathways. Accordingly, TRPC channels have been implicated in a variety of human diseases including cardiovascular disorders such as cardiac hypertrophy, neurodegenerative disorders such as Parkinson&#39;s disease, and neurologic disorders such as spinocerebellar ataxia. Therefore, TRPC channels represent a potential pharmacologic target in human diseases. However, the molecular mechanisms of gating in these channels are still unclear. The difficulty in obtaining large quantities of stable, homogeneous, and purified protein has been a limiting factor in structure determination studies, particularly for mammalian membrane proteins such as the TRPC ion channels. Here, we present a protocol for the large-scale expression of mammalian ion channel membrane proteins using a modified baculovirus gene transfer system and the purification of these proteins by affinity and size-exclusion chromatography. We further present a protocol to collect single-particle cryo-electron microscopy images from purified protein and to use these images to determine the protein structure. Structure determination is a powerful method for understanding the mechanisms of gating and function in ion channels.

This protocol describes techniques used to determine ion channel structures by cryo-electron microscopy, including a baculovirus system used to efficiently express genes in mammalian cells with minimum effort and toxicity, protein extraction, purification, and quality checking, sample grid preparation and screening, as well as data collection and processing.

单粒子冷冻电子显微镜在结构中测定脂质敏感分通道 trpc3 的表达与纯化

Transient receptor potential channels (TRPCs) of the canonical TRP subfamily are nonselective cation channels that play an essential role in calcium ...

Expression, Solubilization, and Purification of Eukaryotic Borate Transporters

The Solute Carrier 4 (SLC4) family of proteins is called the bicarbonate transporters and includes the archetypal protein Anion Exchanger 1 (AE1, also known as Band 3), the most abundant membrane protein in the red blood cells. The SLC4 family is homologous with borate transporters, which have been characterized in plants and fungi. It remains a significant technical challenge to express and purify membrane transport proteins to homogeneity in quantities suitable for&#160;structural or functional studies. Here we describe detailed procedures for the overexpression of borate transporters in Saccharomyces cerevisiae, isolation of yeast membranes, solubilization of protein by detergent, and purification of borate transporter homologs from S. cerevisiae, Arabidopsis thaliana, and Oryza sativa. We also detail a glutaraldehyde cross-linking experiment to assay multimerization of homomeric transporters. Our generalized procedures can be applied to all three proteins and have been optimized for efficacy. Many of the strategies developed here can be utilized for the study of other challenging membrane proteins.

Here we present a protocol to express, solubilize, and purify several eukaryotic borate transporters with homology to the SLC4 transporter family using yeast. We also describe a chemical cross-linking assay to assess the purified homomeric proteins for multimeric assembly. These protocols can be adapted for other challenging membrane proteins.

真核硼酸背液转运体的表达、增溶及纯化

The Solute Carrier 4 (SLC4) family of proteins is called the bicarbonate transporters and includes the archetypal protein Anion Exchanger 1 (AE1, also ...

Expression and Purification of Nuclease-Free Oxygen Scavenger Protocatechuate 3,4-Dioxygenase

Single molecule (SM) microscopy is used in the study of dynamic molecular interactions of fluorophore labeled biomolecules in real time. However, fluorophores are prone to loss of signal via photobleaching by dissolved oxygen (O2). To prevent photobleaching and extend the fluorophore lifetime, oxygen scavenging systems (OSS) are employed to reduce O2. Commercially available OSS may be contaminated by nucleases that damage or degrade nucleic acids, confounding interpretation of experimental results. Here we detail a protocol for the expression and purification of highly active Pseudomonas putida protocatechuate-3,4-dioxygenase (PCD) with no detectable nuclease contamination. PCD can efficiently remove reactive O2 species by conversion of the substrate protocatechuic acid (PCA) to 3-carboxy-cis,cis-muconic acid. This method can be used in any aqueous system where O2 plays a detrimental role in data acquisition. This method is effective in producing highly active, nuclease free PCD in comparison with commercially available PCD.

Protocatechuate 3,4-dioxygenase (PCD) can enzymatically remove free diatomic oxygen from an aqueous system using its substrate protocatechuic acid (PCA). This protocol describes the expression, purification, and activity analysis of this oxygen scavenging enzyme.

无核酸酶的表达和纯化原氧基质 3，4-二氧酶

Single molecule (SM) microscopy is used in the study of dynamic molecular interactions of fluorophore labeled biomolecules in real time. However, ...

Expression, Purification, Crystallization, and Enzyme Assays of Fumarylacetoacetate Hydrolase Domain-Containing Proteins

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this superfamily generally display multi-functionality, involving mainly hydrolase and decarboxylase mechanisms. This article presents a series of consecutive methods for the expression and purification of FAHD proteins, mainly FAHD protein 1 (FAHD1) orthologues among species (human, mouse, nematodes, plants, etc.). Covered methods are protein expression in E. coli, affinity chromatography, ion exchange chromatography, preparative and analytical gel filtration, crystallization, X-ray diffraction, and photometric assays. Concentrated protein of high levels of purity (&#62;98%) may be employed for crystallization or antibody production. Proteins of similar or lower quality may be employed in enzyme assays or used as antigens in detection systems (Western-Blot, ELISA). In the discussion of this work, the identified enzymatic mechanisms of FAHD1 are outlined to describe its hydrolase and decarboxylase bi-functionality in more detail.

Expression and purification of fumarylacetoacetate hydrolase domain-containing proteins is described with examples (expression in E. coli, FPLC). Purified proteins are used for crystallization and antibody production and employed for enzyme assays. Selected photometric assays are presented to display the multi-functionality of FAHD1 as oxaloacetate decarboxylase and acylpyruvate hydrolase.