我们要感谢王卫斯理博士为这项议定书和他宝贵的指导奠定了基础。这项工作得到了国家卫生研究院（授予R01GM127575和R01GM121584）和韦尔奇基金会（授予A-1715）的部分支持。

1. 质粒结构
<ol><li>首先决定哪种高石蛋白将被选中，在哪个赖氨酸部位。使用现场定向突变将网站变异到琥珀色停止胶子 （TAG）。 注:有四个以前设计的质粒用于表达高石蛋白。所有四种高石蛋白都克隆到带有N端结石丁标签的ptDuet-1载体中。Histone H4 还包括一个 SUMO 标签、复制 colE1 的来源，其拷贝编号约为 40，具有抗安眠药和 T7 促进剂。<ol>
<li>设计前向和反向引物，在所需的部位包含 TAG 突变（用琥珀色停止胶子替换现有的 lysine codon）在四个 Histone 蛋白质粒之一。</li>
		<li>使用全质PCR放大包含质粒的标签。确定引号的适当退化温度。一般来说，引体的熔化温度（Tm）为零下5°C就足够了。如果获得 PCR 产品遇到困难，则可以使用 Tm - 5 °C 周围的温度梯度来优化退化温度以进行放大。 注意:在此实验中，内部表示的 PFU 聚合酶用于 30 个周期，条件如下:30 秒 94 °C（变性），30 秒的退化温度，6 分钟 72 °C（或每千基对 1 分钟）。有关此类型的 PCR 方法的更多详细信息，请参阅刘和奈史密斯7。</li>
		<li>通过遵循制造商的协议，使用 PCR 清理套件清理 PCR 产品。通过凝胶电泳和随后的溴化钠染色分析 PCR 产品。</li>
		<li>在 16 °C 的夜间用 T4 韧带孵育，从而对质粒进行石灰。</li>
	</ol>
</li>
	<li>首先将含有质粒（ampR）的标签转换为化学上称职 的大肠杆菌。从转化中挑选至少 4 个菌落并净化质粒。使用标准方法使每个细胞储存甘油，储存在-80°C。 发送测序以确认所需的 TAG 突变。</li>
	<li>一旦序列被确认，使用热冲击法将含标签的质粒 （AmpR）与 pEVOL-AckRS （ChlR）共同转换为具有化学能力的 CobB（ 大肠杆菌中唯一已知的希石裂解乙酰酶）删除 BL21 细胞。pEVOL 是一种质粒，具有中拷贝编号到低拷贝编号 p15A 源。 注:pEVOL系列的质粒是根据先前的研究表明正交aaRS/tRNATyr对的表达增加，有效地减少蛋白质截断和增加突变蛋白8的整体产量。如果 CobB 删除细胞无法访问，则继续使用具有化学能力的 BL21 细胞。CobB是一种类似锡尔图因的石解氨酸脱乙酰酶，在希斯通蛋白表达过程中，可在5mM的最终浓度下添加尼古丁酰胺，以抑制CobB作为替代方法9。</li>
	<li>制作细胞库存，并存储在-80°C。</li>
</ol>2. 甲基基石蛋白表达
<ol><li>在文化烧瓶中准备 1 L 的自动切割 2YT 介质（或选择量）。</li>
	<li>接种 20 mL 的 2YT 介质，其中含有适当的抗生素，并具有含 TAG 的质粒 （AmpR）和 pEVOL-AckRS （ChlR），并在 37 °C 至 OD = 0.6 （4-6 h） 中生长。</li>
	<li>将适当的抗生素添加到 1 L 的自动切割 2YT 介质中，并接种 20 mL 启动器培养物。生长在 37 °C 到 OD = 0.6-0.8 （2-3 小时）。</li>
	<li>将诱导剂和乙酰氨基苯丙胺 （AcK） 添加到 IPTG 1 mM、0.2% 阿拉比诺酶和 AcK 5 mM 的最终浓度中。</li>
	<li>在 37 °C 的 6-8 小时内培养文化。</li>
	<li>15分钟2，700 x g 的颗粒细胞。</li>
	<li>将细胞颗粒存放在-80°C过夜。</li>
	<li>从这一步开始，将样品一直放在冰上。每 1 升培养培养基将细胞颗粒溶解在 50 mL 的希斯通裂解缓冲器中。</li>
	<li>声波根据以下周期:1 s开，1 s关闭，总时间:3分钟在60%振幅。</li>
	<li>颗粒内含体在 41，600 x g 45 分钟。</li>
	<li>丢弃超自然体，并重新将内含物置入 30 mL 的希斯通裂解缓冲器中。颗粒内含物的预兆为 41，600 x g 30 分钟。</li>
	<li>丢弃超自然物，并重新将内含物置入 30 mL 的颗粒清洗缓冲区中。颗粒内含体在 41，600 x g 30 分钟。</li>
	<li>丢弃超自然物，溶解6M盐酸瓜尼丁（GuHCl）缓冲液25mL中的内含体。在 37 °C 的搅拌下孵化 1 小时。 注意:如果需要，包容性机构可以在4°C的夜间与搅拌一起孵育。</li>
	<li>颗粒内含物的预兆为 41，600 x g， 为 45 分钟。用 1 mL 的 Ni-NTA 树脂与 6 M GuHCl 缓冲器平衡 2 小时孵化超自然物。</li>
	<li>在变性条件下进行 Ni-NTA 净化。用 3 列卷的柱洗缓冲器清洗柱。Elute 用 10 mL 的洗脱缓冲器来分型的希石蛋白。 注意:试图在这里浓缩蛋白质是一种选择，但自体酮是非常不可预知的，很容易沉淀。不建议将总容量集中在 2 mL 以上。</li>
	<li>广泛地将乙酰基石蛋白与 5% 醋酸缓冲液对比，以去除盐分。在 4 °C 下一次透析 3 小时，至少换 6 次新鲜缓冲。 注意:为了提高蛋白质纯度，可以选择对纯净水进行透析。然而，这会导致大量降水和产量下降，这可能不利于低产的酸性高蛋白。</li>
	<li>将蛋白质引用并合二为一，并无限期地储存在-80°C。 野生型高石蛋白一般产生10-50毫克/升，而自负的基石蛋白产量低于10毫克/升，具体取决于赖氨酸部位。例如，H3K79Ac 平均为 5 毫克/升。</li>
</ol>3. 野生型石蛋白表达
<ol><li>要组装全核糖体，表达所有4个组蛋白。当表达和净化野生类型的石酮时，协议与自体酮相同，但以下除外:<ol>
<li>不要执行共同转换。不要使用pEVOL-AcKRS进行野生类型的高石表达。相应地调整抗生素。</li>
		<li>在细胞表达的诱导过程中，不要使用CobB删除细胞系或添加尼古丁胺、AcK或阿拉比诺酶。</li>
	</ol>
</li>
</ol>4. 准备601个DNA
注:以前设计的优化DNA序列，以指导核糖体定位组装与高石八度组件，以产生单核体的高效率。此序列称为 601 DNA 或维多姆序列。这个序列已成为核体体外研究的标准DNA序列，从染色质改造检测到单分子测量10。
<ol><li>要为核糖体组装准备大量 601 DNA，将 pGEM-3z/601 转化为前 10 个细胞，并培养 10 mL 的质粒放大和提取。使用质粒提取套件，并遵循制造商的建议。</li>
	<li>使用内部表达的 PFU 聚合酶（更高效）用于此放大协议。设置 PCR 反应:对于 250 μL 反应， 使用 207.5 μL 自动切割 MQ H2O， 25 μL 10x PFU 缓冲区， 5 μL 前进引号: ctggagtggg， 5 μL 反向引言: 阿卡加塔特加卡奇格， 5 μL dNTP 混合， 2.5 μL PFU 酶。我们通常同时运行60个反应，以获得足够的DNA来组装。</li>
	<li>如果使用 PFU 聚合酶，使用 30s 的退化温度为 52 °C，延长时间为 30s。否则按照厂家推荐的条件。</li>
	<li>完成 PCR 后，使用选择的 PCR 清理套件来净化 PCR 产品。</li>
</ol>5. 希斯通八角形的组装
<ol><li>溶解 H2A、H2B、H3 和 H4 中的基石蛋白颗粒，使 GuHCl 缓冲区中每个组蛋白的单独库存量达到 100 μL。</li>
	<li>通过测量 A280 吸收量来计算每个高石蛋白的浓度。</li>
	<li>如果任何蛋白质的吸收大于 1，则用 GuHCl 缓冲器稀释以获得更准确的浓度。</li>
	<li>将 H2A 和 H2B 蛋白质以 1:1 摩尔的比例组合，稀释为总蛋白质浓度为 4 μg/μL。</li>
	<li>隔夜在 4 °C 对 2 M TE 缓冲器时按顺序进行对 1 M TE 缓冲，2 小时以 1 M TE 缓冲，在 5 小时内以 0.5 M TE 缓冲器进行对比。 注:透析的第二步和第三步导致蛋白质的不稳定构象沉淀出来。预计这些步骤将看到强降水。</li>
	<li>在 4 °C 时，在 16，800 x g 时通过离心去除沉淀物。</li>
	<li>再次计算测量A280 吸收度的高石二音量（H2A/H2B混合物）和四合器（H3/H4混合物）的浓度。</li>
	<li>将调光器和四重奏混合在 1:1 摩尔比率中，并通过添加固体 NaCl 将 NaCl 调整为 2 M。</li>
	<li>希斯通八度棉被可存储在4°C，比二十铃和四重奏更稳定。永远不要冻结希斯通八角形，因为这可能导致拆解。</li>
	<li>如果用自体色素组装，只需在程序中用自体蛋白代替野生型蛋白质即可。</li>
</ol>6. 核体组装
<ol><li>使用 100 倍 TE 缓冲器和固体 NaCl 将 601 DNA 调整为 2M TE 缓冲。将 601 DNA 添加到 2M TE 缓冲器中，以 0.85:1 与 0.90:1 的摩尔比加入希斯通八度。如果凝胶移位分析中存在自由DNA，则可以添加较低的脱氧核糖核酸比率。</li>
	<li>将DNA-histone混合物转移到透析袋中，放入约200mL的2M TE缓冲器（或更多，如果组装多个样品），并在4°C时非常轻轻搅拌。</li>
	<li>设置一个永久泵清除，以慢慢滴在没有盐TE缓冲。当音量大约翻倍时，倒出一半的体积。这样做总共至少4次。有了泵，这可能需要4-8小时，这取决于起始体积。当盐浓度降低到150米（用盐度计测量）时，核糖体就会形成。</li>
	<li>盐浓度降低到 150 mM 后，隔夜对 20 mM TE 缓冲器进行透析。</li>
	<li>通过离心去除沉淀物，用读盘器A260 读数测量核糖体的浓度。</li>
	<li>添加他的TEV蛋白酶（TEV:核体1:30，w:w），并在4°C孵育过夜，以去除所有的组胺标签。通过 Ni-NTA 树脂向下拉，从核糖体溶液中去除组胺标签杂质。</li>
	<li>要定位核体并使样品均质化，在 60 °C 孵育 1 小时。 注意:对于特别不稳定的自有网站，此步骤可能不可取。</li>
	<li>将核糖体储存在 4 °C 的短期（几周）。</li>
	<li>对于长期存储，对存储缓冲区进行透析核糖体，并存储在 -80 °C。</li>
</ol>

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任何作者都没有相互竞争的经济利益可以申报。

在实验期间，必须遵循此协议的每一个细节。核糖体不是很稳定，在确定这个协议时经历了太多的反复试验。关键在于在每一步（或观察到时）清除沉淀物，因为颗粒物很容易干扰组装过程。始终将高石样品保存在冰上。如果核糖体在4°C中储存的时间过长，它们可以自发地拆卸。在任何实验中使用之前，如果在 4 °C 中存储超过 2 周，请务必检查本地 PAGE 的任何样本。避免漩涡八度和核体，因为?...

我们利用 PylRS 和 tRNAPyl合成和组装具有特定选型组式的重组单核体。PylRS 作为一种基因密码扩展工具，通过转化后修饰 （PTMs） 生产蛋白质，已被证明是无价的，并且经过基因进化，整合了大约 200 种不同的 ncAAs。PylRS 在琥珀色停止胶子中加入，在翻译过程中消除了其他氨基酸的竞争。PylRS首次在甲基古生物中发现，并自此被用于化学生物学，将新型反应化学组纳入蛋白质1、2。
MmAcKRS1是由甲烷甲基苯丙胺酸盐进化而来，并经常用于我们的实验室合成乙酰化蛋白质3，4，5，6。MmAcKRS1 在埃斯切里西亚大肠杆菌翻译过程中，在首选 mRNA 的琥珀突变站点提供 AcK 。该技术以前曾用于将K4、K9、K14、K18、K23、K27、K36、K56、K64和K79希斯通H3赖氨酸位点的交流电，用于研究Sirtuin 1、2、6和7对乙酰单核体4、5、6的活性。在这里，我们详细介绍了这种表达自体组分子和重组自负核糖体的方法。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.5 M TE Buffer</td>
			<td>NA</td>
			<td>NA</td>
			<td>0.5 M NaCl, 20 mM Tris, 1 mM EDTA, pH 7.8</td>
		</tr>
		<tr>
			<td>1 M TE Buffer</td>
			<td>NA</td>
			<td>NA</td>
			<td>1 M NaCl, 20 mM Tris, 1 mM EDTA, pH 7.8</td>
		</tr>
		<tr>
			<td>100x TE Buffer</td>
			<td>NA</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>2 M TE Buffer</td>
			<td>NA</td>
			<td>NA</td>
			<td>2 M NaCl, 20 mM Tris, 1 mM EDTA, pH 7.8</td>
		</tr>
		<tr>
			<td>20 mM TE Buffer</td>
			<td>NA</td>
			<td>NA</td>
			<td>20 mM NaCl, 20 mM Tris, 1 mM EDTA, pH 7.8</td>
		</tr>
		<tr>
			<td>6 M GuHCl</td>
			<td></td>
			<td></td>
			<td>6M guanidinium chloride, 20 mM Tris, 500 mM NaCl, pH 8.0</td>
		</tr>
		<tr>
			<td>Acetyllysine</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Column Wash Buffer</td>
			<td>NA</td>
			<td>NA</td>
			<td>6 M urea, 500 mM NaCl, 20 mM Tris, 20 mM imidazole pH 7.8</td>
		</tr>
		<tr>
			<td>Elution Buffer</td>
			<td>NA</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand&#160;Variable-Flow Chemical Transfer Pump</td>
			<td>Fischer Scientific</td>
			<td>15-077-67</td>
			<td></td>
		</tr>
		<tr>
			<td>His-TEV protease</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Histone Lysis Buffer</td>
			<td>NA</td>
			<td>NA</td>
			<td>60 mM Tris, 100 mM NaCl, 0.5% Triton-X100 (v/v), 1 mM PMSF pH 8.0</td>
		</tr>
		<tr>
			<td>Ni-NTA Resin</td>
			<td></td>
			<td></td>
			<td>6 M urea, 500 mM NaCl, 20 mM Tris, 250 mM imidazole, pH 7.8</td>
		</tr>
		<tr>
			<td>PCR Clean-Up Kit</td>
			<td>Epoch Life Sicences</td>
			<td>2360050</td>
			<td></td>
		</tr>
		<tr>
			<td>Pellet Wash Buffer</td>
			<td>NA</td>
			<td>NA</td>
			<td>60 mM Tris, 100 mM NaCl, pH 8.0</td>
		</tr>
		<tr>
			<td>petDUET-His-SUMO-TEV-H4</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>petDUET-His-TEV-H2A</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>petDUET-His-TEV-H2B</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>petDUET-His-TEV-H3</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pEVOL-AckRS</td>
			<td>Addgene</td>
			<td>137976</td>
			<td></td>
		</tr>
		<tr>
			<td>pGEM-3z/601</td>
			<td>Addgene</td>
			<td>26656</td>
			<td></td>
		</tr>
		<tr>
			<td>Storage Buffer</td>
			<td>NA</td>
			<td>NA</td>
			<td>20 mM NaCl, 20 mM Tris, 20 mM NaCl, 1 mM EDTA, 20% glycerol, pH 7.8</td>
		</tr>
		<tr>
			<td>Thermocycler</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

site specific lysine acetylation of histones for nucleosome reconstitution using genetic code expansion in escherichia coli

雌二甲酸高石蛋白可以很容易地在 大肠杆菌 编码突变体中表达， Nε- 乙酰赖氨酸 （AcK） 特异性 甲基甲基苯丙胺 -辛泰酶 （MmAcKRS1） 及其同位素 tRNA （tRNAPyl）组装与站点特定乙酰化组蛋白的重组单核体。MmAcKRS1和tRNAPyl 在 埃舍里希亚大肠杆菌翻译过程中，在首选的mRNA的琥珀突变部位提供AcK。该技术已广泛应用于 H3 lysine 站点的 AcK。皮罗利锡-tRNA合成酶（PylRS）也可以很容易地进化，以纳入其他非卡诺氨基酸（ncAAs），用于现场特定的蛋白质修饰或功能化。在这里，我们详细介绍了一种方法，将使用 MmAcKRS1 系统的 AcK 集成到高石 H3 中，并将乙酰化 H3 蛋白质集成到重组后的单核糖体中。Acetylat重组的单核糖体可用于生化和结合测定、结构测定等。获得经过修改的单核糖体对于设计与发现新的相互作用和理解表观遗传学相关的实验至关重要。

通过运行 12% SDS PAGE 凝胶（图 1 和图 2），可以评估调光机、四合器和八角形。在这里你可以看到，一些酸性四合器的产量低于其他（图1）。事实上，离核心区域越近，观察到的产量越低。这很可能是由于乙酰化干扰四聚酯的组装，你离核心区域越近。组装八角形后也观察到类似的影响（?...

Watch this Scientific Journal Video about Site Specific Lysine Acetylation of Histones for Nucleosome Reconstitution using Genetic Code Expansion in Escherichia coli at JoVE.com

Site Specific Lysine Acetylation of Histones for Nucleosome Reconstitution using Genetic Code Expansion in Escherichia coli

在这里，我们提出一种方法来表达使用遗传密码膨胀和组装重组核体体外的自负型高石蛋白。

网站特定赖氨酸乙酰化的组蛋白为核体重组使用遗传密码扩展在 埃斯切里西亚大肠杆菌

Acetylated histone proteins can be easily expressed in Escherichia coli encoding a mutant, N&#949;-acetyl-lysine (AcK)-specific Methanosarcina mazi ...

site-specific-lysine-acetylation-histones-for-nucleosome

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Site Specific Lysine Acetylation of Histones for Nucleosome Reconstitution using Genetic Code Expansion in Escherichia coli

Unit 1

Structure and Organization of DNA

SCIENTISTS IN ACTION

3.9K 次观看.  Texas A&M University. 在这里，我们提出一种方法来表达使用遗传密码膨胀和组装重组核体体外的自负型高石蛋白。

视频: Site Specific Lysine Acetylation of Histones for Nucleosome Reconstitution using Genetic Code Expansion in Escherichia coli

Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification

An increasing interest in applying synthetic biology techniques to program outer membrane vesicles (OMV) are leading to some very interesting and unique applications for OMV where traditional nanoparticles are proving too difficult to synthesize. To date, all Gram-negative bacteria have been shown to produce OMV demonstrating packaging of a variety of cargo that includes small molecules, peptides, proteins and genetic material. Based on their diverse cargo, OMV are implicated in many biological processes ranging from cell-cell communication to gene transfer and delivery of virulence factors depending upon which bacteria are producing the OMV. Only recently have bacterial OMV become accessible for use across a wide range of applications through the development of techniques to control and direct packaging of recombinant proteins into OMV. This protocol describes a method for the production, purification, and use of enzyme packaged OMV providing for improved overall production of recombinant enzyme, increased vesiculation, and enhanced enzyme stability. Successful utilization of this protocol will result in the creation of a bacterial strain that simultaneously produces a recombinant protein and directs it for OMV encapsulation through creating a synthetic linkage between the recombinant protein and an outer membrane anchor protein. This protocol also details methods for isolating OMV from bacterial cultures as well as proper handling techniques and things to consider when adapting this protocol for use for other unique applications such as: pharmaceutical drug delivery, medical diagnostics, and environmental remediation.

Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification

A protocol for the production, purification, and use of enzyme packaged outer membrane vesicles (OMV) providing for enhanced enzyme stability for implementation across diverse applications is presented.

从外膜囊泡内蛋白质导演包装<em&gt;大肠杆菌</em&gt;:设计，生产和纯化

An increasing interest in applying synthetic biology techniques to program outer membrane vesicles (OMV) are leading to some very interesting and unique ...

科研

生物化学

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

Bacteria are frequently exposed to environmental changes, such as alterations in pH, temperature, redox status, light exposure or mechanical force. Many of these conditions cause protein unfolding in the cell and have detrimental impact on the survival of the organism. A group of unrelated, stress-specific molecular chaperones have been shown to play essential roles in the survival of these stress conditions. While fully folded and chaperone-inactive before stress, these proteins rapidly unfold and become chaperone-active under specific stress conditions. Once activated, these conditionally disordered chaperones bind to a large number of different aggregation-prone proteins, prevent their aggregation and either directly or indirectly facilitate protein refolding upon return to non-stress conditions. The primary approach for gaining a more detailed understanding about the mechanism of their activation and client recognition involves the purification and subsequent characterization of these proteins using in vitro chaperone assays. Follow-up in vivo stress assays are absolutely essential to independently confirm the obtained in vitro results.
This protocol describes in vitro and in vivo methods to characterize the chaperone activity of E. coli HdeB, an acid-activated chaperone. Light scattering measurements were used as a convenient read-out for HdeB&#39;s capacity to prevent acid-induced aggregation of an established model client protein, MDH, in vitro. Analytical ultracentrifugation experiments were applied to reveal complex formation between HdeB and its client protein LDH, to shed light into the fate of client proteins upon their return to non-stress conditions. Enzymatic activity assays of the client proteins were conducted to monitor the effects of HdeB on pH-induced client inactivation and reactivation. Finally, survival studies were used to monitor the influence of HdeB&#39;s chaperone function in vivo.

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

This study describes biophysical, biochemical and molecular techniques to characterize the chaperone activity of Escherichia coli HdeB under acidic pH conditions. These methods have been successfully applied for other acid-protective chaperones such as HdeA and can be modified to work for other chaperones and stress conditions.

的pH依赖性活性的检测<em&gt;大肠杆菌</em&gt;陪护HdeB<em&gt;体外</em&gt;和<em&gt;在体内</em

Bacteria are frequently exposed to environmental changes, such as alterations in pH, temperature, redox status, light exposure or mechanical force. Many ...

Efficient and Site-specific Antibody Labeling by Strain-promoted Azide-alkyne Cycloaddition

There are currently many chemical tools available to introduce chemical probes into proteins to study their structure and function. A useful method is protein conjugation by genetically introducing an unnatural amino acid containing a bioorthogonal functional group. This report describes a detailed protocol for site-specific antibody conjugation. The protocol includes experimental details for the genetic incorporation of an azide-containing amino acid, and the conjugation reaction by strain-promoted azide-alkyne cycloaddition (SPAAC). This strain-promoted reaction proceeds by simple mixing of the reacting molecules at physiological pH and temperature, and does not require additional reagents such as copper(I) ions and copper-chelating ligands. Therefore, this method would be useful for general protein conjugation and development of antibody drug conjugates (ADCs).

Here, we present a protocol to site-specifically introduce chemical probes into an antibody fragment by genetically incorporating an azide-containing amino acid, and subsequently coupling the azide with a chemical probe by strain-promoted azide-alkyne cycloaddition (SPAAC).

高效抗体标记由应变叠氮推广 - 炔环加成特定站点

There are currently many chemical tools available to introduce chemical probes into proteins to study their structure and function. A useful method is ...

Reconstitution of Nucleosomes with Differentially Isotope-labeled Sister Histones

Asymmetrically modified nucleosomes contain the two copies of a histone (sister histones) decorated with distinct sets of Post-translational Modifications (PTMs). They are newly identified species with unknown means of establishment and functional implications. Current analytical methods are inadequate to detect the copy-specific occurrence of PTMs on the nucleosomal sister histones. This protocol presents a biochemical method for the in vitro reconstitution of nucleosomes containing differentially isotope-labeled sister histones. The generated complex can be also asymmetrically modified, after including a premodified histone pool during refolding of histone subcomplexes. These asymmetric nucleosome preparations can be readily reacted with histone-modifying enzymes to study modification cross-talk mechanisms imposed by the asymmetrically pre-incorporated PTM using nuclear magnetic resonance (NMR) spectroscopy. Particularly, the modification reactions in real-time can be mapped independently on the two sister histones by performing different types of NMR correlation experiments, tailored for the respective isotope type. This methodology provides the means to study crosstalk mechanisms that contribute to the formation and propagation of asymmetric PTM patterns on nucleosomal complexes.

This protocol describes the reconstitution of nucleosomes containing differentially isotope-labeled sister histones. At the same time, asymmetrically post-translationally modified nucleosomes can be generated after using a premodified histone copy. These preparations can be readily used to study modification crosstalk mechanisms, simultaneously on both sister histones, by using high-resolution NMR spectroscopy.

核小体的重建与差异同位素标记的姐妹组蛋白

Asymmetrically modified nucleosomes contain the two copies of a histone (sister histones) decorated with distinct sets of Post-translational Modifications ...

Quantification of the Abundance and Charging Levels of Transfer RNAs in Escherichia coli

Transfer RNA (tRNA) is an essential part of the translational machinery in any organism. tRNAs bind and transfer amino acids to the translating ribosome. The relative levels of different tRNAs, and the ratio of aminoacylated tRNA to total tRNA, known as the charging level, are important factors in determining the accuracy and speed of translation. Therefore, the abundance and charging levels of tRNAs are important variables to measure when studying protein synthesis, for example under various stress conditions. Here, we describe a method for harvesting tRNA and directly measuring both the relative abundance and the absolute charging level of specific tRNA species in Escherichia coli. The tRNA is harvested in such a way that the labile bond between the tRNA and its amino acid is preserved. The RNA is then subjected to gel electrophoresis and Northern blotting, which results in separation of the charged and uncharged tRNAs. The levels of specific tRNAs in different samples can be compared due to the addition of spike-in cells for normalization. Prior to RNA purification, we add 5% of E. coli cells that overproduce the rare tRNAselC to each sample. The amount of the tRNA species of interest in a sample is then normalized to the amount of tRNAselC in the same sample. Addition of spike-in cells prior to RNA purification has the advantage over addition of purified spike-in RNAs that it also accounts for any differences in cell lysis efficiency between samples.

Quantification of the Abundance and Charging Levels of Transfer RNAs in Escherichia coli

Here we present a method for directly measuring transfer RNA charging levels from purified Escherichia coli RNA as well as a way to compare relative levels of transfer RNA, or any other short RNA, across different samples based on the addition of spike-in cells expressing a reference gene.

丰度和收费水平的转移 Rna 在大肠杆菌中的量化

Transfer RNA (tRNA) is an essential part of the translational machinery in any organism. tRNAs bind and transfer amino acids to the translating ribosome. ...

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 ...

Large-scale Production of Recombinant RNAs on a Circular Scaffold Using a Viroid-derived System in Escherichia coli

With increasing interest in RNA biology and the use of RNA molecules in sophisticated biotechnological applications, the methods to produce large amounts of recombinant RNAs are limited. Here, we describe a protocol to produce large amounts of recombinant RNA in Escherichia coli based on co-expression of a chimeric molecule that contains the RNA of interest in a viroid scaffold and a plant tRNA ligase. Viroids are relatively small, non-coding, highly base-paired circular RNAs that are infectious to higher plants. The host plant tRNA ligase is an enzyme recruited by viroids that belong to the family Avsunviroidae, such as Eggplant latent viroid (ELVd), to mediate RNA circularization during viroid replication. Although ELVd does not replicate in E. coli, an ELVd precursor is efficiently transcribed by the E. coli RNA polymerase and processed by the embedded hammerhead ribozymes in bacterial cells, and the resulting monomers are circularized by the co-expressed tRNA ligase reaching a remarkable concentration. The insertion of an RNA of interest into the ELVd scaffold enables the production of tens of milligrams of the recombinant RNA per liter of bacterial culture in regular laboratory conditions. A main fraction of the RNA product is circular, a feature that facilitates the purification of the recombinant RNA to virtual homogeneity. In this protocol, a complementary DNA (cDNA) corresponding to the RNA of interest is inserted in a particular position of the ELVd cDNA in an expression plasmid that is used, along the plasmid to co-express eggplant tRNA ligase, to transform E. coli. Co-expression of both molecules under the control of strong constitutive promoters leads to production of large amounts of the recombinant RNA. The recombinant RNA can be extracted from the bacterial cells and separated from the bulk of bacterial RNAs taking advantage of its circularity.

Large-scale Production of Recombinant RNAs on a Circular Scaffold Using a Viroid-derived System in Escherichia coli

Here, we present a protocol to produce large amounts of recombinant RNA in Escherichia coli by co-expressing a chimeric RNA that contains the RNA of interest in a viroid scaffold and a plant tRNA ligase. The main product is a circular molecule that facilitates purification to homogeneity.

利用病毒衍生系统在大肠杆菌中大规模生产圆形脚手架上的重组 rna

With increasing interest in RNA biology and the use of RNA molecules in sophisticated biotechnological applications, the methods to produce large amounts ...

Site-Directed Mutagenesis for In Vitro and In Vivo Experiments Exemplified with RNA Interactions in Escherichia Coli

Site-directed mutagenesis is a technique used to introduce specific mutations in DNA to investigate the interaction between small non-coding ribonucleic acid (sRNA) molecules and target messenger RNAs (mRNAs). In addition, site-directed mutagenesis is used to map specific protein binding sites to RNA. A 2-step and 3-step PCR based introduction of mutations is described. The approach is relevant to all protein-RNA and RNA-RNA interaction studies. In short, the technique relies on designing primers with the desired mutation(s), and through 2 or 3 steps of PCR synthesizing a PCR product with the mutation. The PCR product is then used for cloning. Here, we describe how to perform site-directed mutagenesis with both the 2- and 3-step approach to introduce mutations to the sRNA, McaS, and the mRNA, csgD, to investigate RNA-RNA and RNA-protein interactions. We apply this technique to investigate RNA interactions; however, the technique is applicable to all mutagenesis studies (e.g., DNA-protein interactions, amino-acid substitution/deletion/addition). It is possible to introduce any kind of mutation except for non-natural bases but the technique is only applicable if a PCR product can be used for downstream application (e.g., cloning and template for further PCR).

Site-Directed Mutagenesis for In Vitro and In Vivo Experiments Exemplified with RNA Interactions in Escherichia Coli

Site-directed mutagenesis is a technique used to introduce specific mutations in deoxyribonucleic acid (DNA). This protocol describes how to do site-directed mutagenesis with a 2-step and 3-step polymerase chain reaction (PCR) based approach, which is applicable to any DNA fragment of interest.

体外和体内实验的位定向诱变-以大肠杆菌rna相互作用为例

Site-directed mutagenesis is a technique used to introduce specific mutations in DNA to investigate the interaction between small non-coding ribonucleic ...

In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones

Ubiquitination is a post-translational modification that plays important roles in various signaling pathways and is notably involved in the coordination of chromatin function and DNA-associated processes. This modification involves a sequential action of several enzymes including E1 ubiquitin-activating, E2 ubiquitin-conjugating and E3 ubiquitin-ligase and is reversed by deubiquitinases (DUBs). Ubiquitination induces degradation of proteins or alteration of protein function including modulation of enzymatic activity, protein-protein interaction and subcellular localization. A critical step in demonstrating protein ubiquitination or deubiquitination is to perform in vitro reactions with purified components. Effective ubiquitination and deubiquitination reactions could be greatly impacted by the different components used, enzyme co-factors, buffer conditions, and the nature of the substrate.&#160; Here, we provide step-by-step protocols for conducting ubiquitination and deubiquitination reactions. We illustrate these reactions using minimal components of the mouse Polycomb Repressive Complex 1 (PRC1), BMI1, and RING1B, an E3 ubiquitin ligase that monoubiquitinates histone H2A on lysine 119. Deubiquitination of nucleosomal H2A is performed using a minimal Polycomb Repressive Deubiquitinase (PR-DUB) complex formed by the human deubiquitinase BAP1 and the DEUBiquitinase ADaptor (DEUBAD) domain of its co-factor ASXL2. These ubiquitination/deubiquitination assays can be conducted in the context of either recombinant nucleosomes reconstituted with bacteria-purified proteins or native nucleosomes purified from mammalian cells. We highlight the intricacies that can have a significant impact on these reactions and we propose that the general principles of these protocols can be swiftly adapted to other E3 ubiquitin ligases and deubiquitinases.

Ubiquitination is a post-translational modification that plays important roles in cellular processes and is tightly coordinated by deubiquitination. Defects in both reactions underlie human pathologies. We provide protocols for conducting ubiquitination and deubiquitination reaction in vitro using purified components.

核糖体组蛋白的体外泛化和二次基质测定

Ubiquitination is a post-translational modification that plays important roles in various signaling pathways and is notably involved in the coordination ...

Extraction and Visualization of Protein Aggregates after Treatment of Escherichia coli with a Proteotoxic Stressor

The exposure of living organisms to environmental and cellular stresses often causes disruptions in protein homeostasis and can result in protein aggregation. The accumulation of protein aggregates in bacterial cells can lead to significant alterations in the cellular phenotypic behavior, including a reduction in growth rates, stress resistance, and virulence. Several experimental procedures exist for the examination of these stressor-mediated phenotypes. This paper describes an optimized assay for the extraction and visualization of aggregated and soluble proteins from different Escherichia coli strains after treatment with a silver-ruthenium-containing antimicrobial. This compound is known to generate reactive oxygen species and causes widespread protein aggregation. 
The method combines a centrifugation-based separation of protein aggregates and soluble proteins from treated and untreated cells with subsequent separation and visualization by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie staining. This approach is simple, fast, and allows a qualitative comparison of protein aggregate formation in different E. coli strains. The methodology has a wide range of applications, including the possibility to investigate the impact of other proteotoxic antimicrobials on in vivo protein aggregation in a wide range of bacteria. Moreover, the protocol can be used to identify genes that contribute to increased resistance to proteotoxic substances. Gel bands can be used for the subsequent identification of proteins that are particularly prone to aggregation.

Extraction and Visualization of Protein Aggregates after Treatment of Escherichia coli with a Proteotoxic Stressor

This protocol describes the extraction and visualization of aggregated and soluble proteins from Escherichia coli after treatment with a proteotoxic antimicrobial. Following this procedure allows a qualitative comparison of protein aggregate formation in vivo in different bacterial strains and/or between treatments.

网站特定赖氨酸乙酰化的组蛋白为核体重组使用遗传密码扩展在 埃斯切里西亚大肠杆菌

网站特定赖氨酸乙酰化的组蛋白为核体重组使用遗传密码扩展在埃斯切里西亚大肠杆菌