Name: site specific lysine acetylation of histones for nucleosome reconstitution using genetic code expansion in escherichia coli
Uploaded: 2020-12-26T14:00:00.000+00:00
Duration: 7 min 26 s
Description: 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

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>

<ol>
	<li>Srinivasan, G., James, C. M., Krzycki, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pyrrolysine+encoded+by+UAG+in+Archaea:+Charging+of+a+UAG-Decoding+specialized+tRNA.">Pyrrolysine encoded by UAG in Archaea: Charging of a UAG-Decoding specialized tRNA.</a> Science. 296 (5572), 1459-1462 (2002).</li><li>Wan, W., Tharp, J. M., Liu, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pyrrolysyl-tRNA+synthetase:+an+ordinary+enzyme+but+an+outstanding+genetic+code+expansion+tool.">Pyrrolysyl-tRNA synthetase: an ordinary enzyme but an outstanding genetic code expansion tool.</a> Biochimica Et Biophysica Acta. 1844 (6), 1059-1070 (2014).</li><li>Umehara, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N-Acetyl+lysyl-tRNA+synthetases+evolved+by+a+CcdB-based+selection+possess+N-acetyl+lysine+specificity+in+vitro+and+in+vivo.">N-Acetyl lysyl-tRNA synthetases evolved by a CcdB-based selection possess N-acetyl lysine specificity in vitro and in vivo.</a> FEBS Letters. 586 (6), 729-733 (2012).</li><li>Hsu, W. W., Wu, B., Liu, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sirtuins+1+and+2+are+universal+histone+deacetylases.">Sirtuins 1 and 2 are universal histone deacetylases.</a> ACS Chemical Biology. 11 (3), 792-799 (2016).</li><li>Wang, W. W., Zeng, Y., Wu, B., Deiters, A., Liu, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+chemical+biology+approach+to+reveal+Sirt6-targeted+histone+H3+sites+in+nucleosomes.">A chemical biology approach to reveal Sirt6-targeted histone H3 sites in nucleosomes.</a> ACS Chemical Biology. 11 (7), 1973-1981 (2016).</li><li>Wang, W. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+click+chemistry+approach+reveals+the+chromatin-dependent+histone+H3K36+deacylase+nature+of+SIRT7.">A click chemistry approach reveals the chromatin-dependent histone H3K36 deacylase nature of SIRT7.</a> Journal of the American Chemical Society. 141 (6), 2462-2473 (2019).</li><li>Liu, H., Naismith, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+efficient+one-step+site-directed+deletion,+insertion,+single+and+multiple-site+plasmid+mutagenesis+protocol.">An efficient one-step site-directed deletion, insertion, single and multiple-site plasmid mutagenesis protocol.</a> BMC Biotechnology. 8 (1), 91 (2008).</li><li>Young, T. S., Ahmad, I., Yin, J. A., Schultz, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+enhanced+system+for+unnatural+amino+acid+mutagenesis+in+E.+coli.">An enhanced system for unnatural amino acid mutagenesis in E. coli.</a> Journal of Molecular Biology. 395 (2), 361-374 (2010).</li><li>Gallego-Jara, J., &#201;cija Conesa, A., de Diego Puente, T., Lozano Terol, G., C&#225;novas D&#237;az, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+CobB+kinetics+and+inhibition+by+nicotinamide.">Characterization of CobB kinetics and inhibition by nicotinamide.</a> PLoS One. 12 (12), 0189689 (2017).</li><li>Lowary, P. T., Widom, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+DNA+sequence+rules+for+high+affinity+binding+to+histone+octamer+and+sequence-directed+nucleosome+positioning.">New DNA sequence rules for high affinity binding to histone octamer and sequence-directed nucleosome positioning.</a> Journal of Molecular Biology. 276 (1), 19-42 (1998).</li><li>Zhao, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+basis+of+tight+nuclear+tethering+and+inactivation+of+cGAS.">The molecular basis of tight nuclear tethering and inactivation of cGAS.</a> Nature. 587 (7835), 673-677 (2020).</li><li>Burdette, D. L., Vance, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=STING+and+the+innate+immune+response+to+nucleic+acids+in+the+cytosol.">STING and the innate immune response to nucleic acids in the cytosol.</a> Nature Immunology. 14 (1), 19-26 (2013).</li><li>Barber, G. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innate+immune+DNA+sensing+pathways:+STING,+AIMII+and+the+regulation+of+interferon+production+and+inflammatory+responses.">Innate immune DNA sensing pathways: STING, AIMII and the regulation of interferon production and inflammatory responses.</a> Current Opinion in Immunology. 23 (1), 10-20 (2011).</li><li>Ablasser, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=cGAS+produces+a+2'-5'-linked+cyclic+dinucleotide+second+messenger+that+activates+STING.">cGAS produces a 2'-5'-linked cyclic dinucleotide second messenger that activates STING.</a> Nature. 498 (7454), 380-384 (2013).</li><li>Gao, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclic+[G(2',5')pA(3',5')p]+is+the+metazoan+second+messenger+produced+by+DNA-activated+cyclic+GMP-AMP+synthase.">Cyclic [G(2',5')pA(3',5')p] is the metazoan second messenger produced by DNA-activated cyclic GMP-AMP synthase.</a> Cell. 153 (5), 1094-1107 (2013).</li><li>Zhao, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+conserved+PLPLRT/SD+motif+of+STING+mediates+the+recruitment+and+activation+of+TBK1.">A conserved PLPLRT/SD motif of STING mediates the recruitment and activation of TBK1.</a> Nature. 569 (7758), 718-722 (2019).</li></ol>

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><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&amp;nbsp;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

Universidad de Cartagena UDEC

Research

Education

JoVE Journal

JoVE Core

Cell Biology

Biochemistry

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

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 ...

科研

生物化学

Analysis of Histone Antibody Specificity with Peptide Microarrays

Post-translational modifications (PTMs) on histone proteins are widely studied for their roles in regulating chromatin structure and gene expression. The mass production and distribution of antibodies specific to histone PTMs has greatly facilitated research on these marks. As histone PTM antibodies are key reagents for many chromatin biochemistry applications, rigorous analysis of antibody specificity is necessary for accurate data interpretation and continued progress in the field. This protocol describes an integrated pipeline for the design, fabrication and use of peptide microarrays for profiling the specificity of histone antibodies. The design and analysis aspects of this procedure are facilitated by ArrayNinja, an open-source and interactive software package we recently developed to streamline the customization of microarray print formats. This pipeline has been used to screen a large number of commercially available and widely used histone PTM antibodies, and data generated from these experiments are freely available through an online and expanding Histone Antibody Specificity Database. Beyond histones, the general methodology described herein can be applied broadly to the analysis of PTM-specific antibodies.

This manuscript describes methods for applying peptide microarray technology to specificity profiling of antibodies that recognize histones and their post-translational modifications.

用肽微阵列分析组蛋白抗体特异性

Post-translational modifications (PTMs) on histone proteins are widely studied for their roles in regulating chromatin structure and gene expression. The ...

Optimizing the Genetic Incorporation of Chemical Probes into GPCRs for Photo-crosslinking Mapping and Bioorthogonal Chemistry in Live Mammalian Cells

The genetic incorporation of non-canonical amino acids (ncAAs) via amber stop codon suppression is a powerful technique to install artificial probes and reactive moieties onto proteins directly in the live cell. Each ncAA is incorporated by a dedicated orthogonal suppressor-tRNA/amino-acyl-tRNA-synthetase (AARS) pair that is imported into the host organism. The incorporation efficiency of different ncAAs can greatly differ, and be unsatisfactory in some cases. Orthogonal pairs can be improved by manipulating either the AARS or the tRNA. However, directed evolution of tRNA or AARS using large libraries and dead/alive selection methods are not feasible in mammalian cells. Here, a facile and robust fluorescence-based assay to evaluate the efficiency of orthogonal pairs in mammalian cells is presented. The assay allows screening tens to hundreds of AARS/tRNA variants with a moderate effort and within a reasonable time. Use of this assay to generate new tRNAs that significantly improve the efficiency of the pyrrolysine orthogonal system is described, along with the application of ncAAs to the study of G-protein coupled receptors (GPCRs), which are challenging objects for ncAA mutagenesis. First, by systematically incorporating a photo-crosslinking ncAA throughout the extracellular surface of a receptor, binding sites of different ligands on the intact receptor are mapped directly in the live cell. Second, by incorporating last-generation ncAAs into a GPCR, ultrafast catalyst-free receptor labeling with a fluorescent dye is demonstrated, which exploits bioorthogonal strain-promoted inverse Diels Alder cycloaddition (SPIEDAC) on the live cell. As ncAAs can be generally applied to any protein independently on its size, the method is of general interest for a number of applications. In addition, ncAA incorporation does not require any special equipment and is easily performed in standard biochemistry labs.

A facile fluorescence assay is presented to evaluate the efficiency of amino-acyl-tRNA-synthetase/tRNA pairs incorporating non-canonical amino-acids (ncAAs) into proteins expressed in mammalian cells. The application of ncAAs to study G-protein coupled receptors (GPCRs) is described, including photo-crosslinking mapping of binding sites and bioorthogonal GPCR labeling on live cells.

生物哺乳动物细胞光交联图谱和 Bioorthogonal 化学中化学探针的遗传受体优化

The genetic incorporation of non-canonical amino acids (ncAAs) via amber stop codon suppression is a powerful technique to install artificial probes and ...

化学

Detection of Post-translational Modifications on Native Intact Nucleosomes by ELISA

The genome of eukaryotes exists as chromatin which contains both DNA and proteins. The fundamental unit of chromatin is the nucleosome, which contains 146 base pairs of DNA associated with two each of histones H2A, H2B, H3, and H41. The N-terminal tails of histones are rich in lysine and arginine and are modified post-transcriptionally by acetylation, methylation, and other post-translational modifications (PTMs). The PTM configuration of nucleosomes can affect the transcriptional activity of associated DNA, thus providing a mode of gene regulation that is epigenetic in nature 2,3. We developed a method called nucleosome ELISA (NU-ELISA) to quantitatively determine global PTM signatures of nucleosomes extracted from cells. NU-ELISA is more sensitive and quantitative than western blotting, and is useful to interrogate the epiproteomic state of specific cell types. This video journal article shows detailed procedures to perform NU-ELISA analysis.

Nucleosome ELISA (NU-ELISA) is a sensitive and quantitative method to detect global patterns of post-translational modifications in preparations of native, intact nucleosomes. These modifications include methylations, acetylations, and phosphorylations at specific histone amino acid residues, and hence NU-ELISA provides a global proteomic assay of the overall chromatin modification states of specific cell types.

检测采用ELISA法的母语完整的核小体的翻译后修饰

The genome of eukaryotes exists as chromatin which contains both DNA and proteins. The fundamental unit of chromatin is the nucleosome, which contains 146 ...

生物学

Repressing Gene Transcription by Redirecting Cellular Machinery with Chemical Epigenetic Modifiers

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene expression regulation are commonly disrupted in many diseases, a locus-specific, endogenous, and reversible method to study and control these mechanisms of action has been lacking. To address this issue, we have developed and characterized novel gene-regulating bifunctional molecules. One component of the bifunctional molecule binds to a DNA-protein anchor so that it will be recruited to an allele-specific locus. The other component engages endogenous cellular chromatin-modifying machinery, recruiting these proteins to a gene of interest. These small molecules, called chemical epigenetic modifiers (CEMs), are capable of controlling gene expression and the chromatin environment in a dose-dependent and reversible manner. Here, we detail a CEM approach and its application to decrease gene expression and histone tail acetylation at a Green Fluorescent Protein (GFP) reporter located at the Oct4 locus in mouse embryonic stem cells (mESCs). We characterize the lead CEM (CEM23) using fluorescent microscopy, flow cytometry, and chromatin immunoprecipitation (ChIP), followed by a quantitative polymerase chain reaction (qPCR). While the power of this system is demonstrated at the Oct4 locus, conceptually, the CEM technology is modular and can be applied in other cell types and at other genomic loci.

Regulation of the chromatin environment is an essential process required for proper gene expression. Here, we describe a method for controlling gene expression through the recruitment of chromatin-modifying machinery in a gene-specific and reversible manner.

用化学表观修饰因子重定向细胞机械抑制基因转录

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene ...

生物工程

<meta charset="utf8"></head><body>用于单分子研究的原位核小体重构

In Situ Nucleosome Assembly for Single-Molecule Correlative Force and Fluorescence Microscopy

Nucleosomes constitute the primary unit of eukaryotic chromatin and have been the focus of numerous informative single-molecule investigations regarding their biophysical properties and interactions with chromatin-binding proteins. Nucleosome reconstitution on DNA for these studies typically involves a salt dialysis procedure that provides precise control over the placement and number of nucleosomes formed along a DNA tether. However, this protocol is time-consuming and requires a substantial amount of DNA and histone octamers as inputs. To offer an alternative strategy, an in situ nucleosome reconstitution method for single-molecule force and fluorescence microscopy that utilizes the histone chaperone Nap1 is described. This method enables users to assemble nucleosomes on any DNA template without the need for strong nucleosome positioning sequences, adjust nucleosome density on demand, and use fewer reagents. In situ nucleosome formation occurs within seconds, offering a simpler experimental workflow and a convenient transition into single-molecule measurements. Examples of two downstream assays for probing nucleosome mechanics and visualizing the behavior of individual proteins on chromatin are further described.

<meta charset="utf8"></head><body>作者聚焦：用于单分子技术的高效核小体重构

This article presents a detailed experimental procedure for reconstituting nucleosome-containing DNA tethers for single-molecule correlative force and fluorescence microscopy. It further describes several downstream experiments that can be conducted to visualize the binding behavior of chromatin-interacting proteins and analyze changes in the physical properties of nucleosomes.

用于单分子相关力和荧光显微镜的原位核小体组装

Nucleosomes constitute the primary unit of eukaryotic chromatin and have been the focus of numerous informative single-molecule investigations regarding ...

A Facile Protocol to Generate Site-Specifically Acetylated Proteins in Escherichia Coli

Post-translational modifications that occur at specific positions of proteins have been shown to play important roles in a variety of cellular processes. Among them, reversible lysine acetylation is one of the most widely distributed in all domains of life. Although numerous mass spectrometry-based acetylome studies have been performed, further characterization of these putative acetylation targets has been limited. One possible reason is that it is difficult to generate purely acetylated proteins at desired positions by most classic biochemical approaches. To overcome this challenge, the genetic code expansion technique has been applied to use the pair of an engineered pyrrolysyl-tRNA synthetase variant, and its cognate tRNA from Methanosarcinaceae species, to direct the cotranslational incorporation of acetyllysine at the specific site in the protein of interest. After first application in the study of histone acetylation, this approach has facilitated acetylation studies on a variety of proteins. In this work, we demonstrated a facile protocol to produce site-specifically acetylated proteins by using the model bacterium Escherichia coli as the host. Malate dehydrogenase was used as a demonstration example in this work.

A Facile Protocol to Generate Site-Specifically Acetylated Proteins in Escherichia Coli

Genetic code expansion serves as a powerful tool to study a wide range of biological processes, including protein acetylation. Here we demonstrate a facile protocol to exploit this technique for generating homogeneously acetylated proteins at specific sites in Escherichia coli cells.

在大肠杆菌中生成特异的乙酰蛋白的简易协议

Post-translational modifications that occur at specific positions of proteins have been shown to play important roles in a variety of cellular processes. ...

免疫与感染

Application of MassSQUIRM for Quantitative Measurements of Lysine Demethylase Activity

Recently, epigenetic regulators have been discovered as key players in many different diseases 1-3. As a result, these enzymes are prime targets for small molecule studies and drug development 4. Many epigenetic regulators have only recently been discovered and are still in the process of being classified. Among these enzymes are lysine demethylases which remove methyl groups from lysines on histones and other proteins. Due to the novel nature of this class of enzymes, few assays have been developed to study their activity. This has been a road block to both the classification and high throughput study of histone demethylases. Currently, very few demethylase assays exist. Those that do exist tend to be qualitative in nature and cannot simultaneously discern between the different lysine methylation states (un-, mono-, di- and tri-). Mass spectrometry is commonly used to determine demethylase activity but current mass spectrometric assays do not address whether differentially methylated peptides ionize differently. Differential ionization of methylated peptides makes comparing methylation states difficult and certainly not quantitative (Figure 1A). Thus available assays are not optimized for the comprehensive analysis of demethylase activity.
Here we describe a method called MassSQUIRM (mass spectrometric quantitation using isotopic reductive methylation) that is based on reductive methylation of amine groups with deuterated formaldehyde to force all lysines to be di-methylated, thus making them essentially the same chemical species and therefore ionize the same (Figure 1B). The only chemical difference following the reductive methylation is hydrogen and deuterium, which does not affect MALDI ionization efficiencies. The MassSQUIRM assay is specific for demethylase reaction products with un-, mono- or di-methylated lysines. The assay is also applicable to lysine methyltransferases giving the same reaction products. Here, we use a combination of reductive methylation chemistry and MALDI mass spectrometry to measure the activity of LSD1, a lysine demethylase capable of removing di- and mono-methyl groups, on a synthetic peptide substrate 5. This assay is simple and easily amenable to any lab with access to a MALDI mass spectrometer in lab or through a proteomics facility. The assay has ~8-fold dynamic range and is readily scalable to plate format 5.

We present a method for using MALDI mass spectrometry and reductive methylation chemistry to quantify changes in lysine methylation.

赖氨酸去甲基化酶活性的定量测量中的应用的MassSQUIRM

Recently, epigenetic regulators have been discovered as key players in many different diseases 1-3. As a result, these enzymes are prime targets for small ...

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

摘要

探索更多视频

此视频中的章节

核小体的重建与差异同位素标记的姐妹组蛋白

用肽微阵列分析组蛋白抗体特异性

生物哺乳动物细胞光交联图谱和 Bioorthogonal 化学中化学探针的遗传受体优化

检测采用ELISA法的母语完整的核小体的翻译后修饰

用化学表观修饰因子重定向细胞机械抑制基因转录

用于单分子相关力和荧光显微镜的原位核小体组装

用于单分子相关力和荧光显微镜的原位核小体组装

用于单分子相关力和荧光显微镜的原位核小体组装

在大肠杆菌中生成特异的乙酰蛋白的简易协议

赖氨酸去甲基化酶活性的定量测量中的应用的MassSQUIRM

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

摘要

探索更多视频

此视频中的章节

核小体的重建与差异同位素标记的姐妹组蛋白

用肽微阵列分析组蛋白抗体特异性

生物哺乳动物细胞光交联图谱和 Bioorthogonal 化学中化学探针的遗传受体优化

检测采用ELISA法的母语完整的核小体的翻译后修饰

用化学表观修饰因子重定向细胞机械抑制基因转录

用于单分子相关力和荧光显微镜的原位核小体组装

用于单分子相关力和荧光显微镜的原位核小体组装

用于单分子相关力和荧光显微镜的原位核小体组装

在大肠杆菌中生成特异的乙酰蛋白的简易协议

赖氨酸去甲基化酶活性的定量测量中的应用的MassSQUIRM

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