这项工作得到了国立卫生研究院 (AI119813) 的支持, 这是来自阿肯色州大学的起步, 也是来自阿肯色州生物科学研究所的奖项。

1. 靶基因的定点诱变
注意: MDH 在pCDF-1向量中以CloDF13原点和20到 4034的副本号表示在 T7 启动子下。
<ol><li>介绍了琥珀色停止密码在该基因的位置140的引物 (向前底漆: GGTGTTTATGAC标记AACAAACTGTTCGGCG 和反向底漆: GGCTTTTTTCAGCACTTCAGCAGCAATTGC), 按照指示的网站定向诱变试剂盒。</li>
	<li>放大含有野生型苹果脱水基因的模板质粒, 通过聚合酶链反应 (PCR) 反应, 插入停止密码子突变。在反应混合物中, 包括12.5 µL 的 2X DNA 聚合酶组合物, 1.25 µL 10 µM 前引物, 1.25 µL 10 µM 反转底漆, 1 µL 模板 DNA (20 ng/µL) (pCDF-1质粒含有野生型 MDH 的基因), 9 µL 无核酸水。<ol>
<li>使用 PCR 反应参数如下: 三十年代初变性在98° c;25周期十年代在98° c, 三十年代在55° c 和 3 min 在72° c;最后的引伸在72° c 为 3 min。PCR 后, 将放大的材料直接添加到激酶-连接-DpnI 酶混合从试剂盒1小时的室温 circularization 和模板去除。 注: 反应混合物含有1µL PCR 产物, 5 µL 2X 反应缓冲, 1 µL 10X 激酶连接 DpnI 酶混合, 3 µL 无核酸水。</li>
	</ol>
</li>
	<li>添加5µL 的反应混合物的管25µL 解冻合格的大肠杆菌DH5α细胞从试剂盒。小心地弹管混合, 并将混合物放在冰上30分钟. 热冲击混合物在42° c 为三十年代和地方在冰为另外的5分钟。<ol>
<li>吸管600µL 室温超优化汤与物抑制 (SOC) 介质从试剂盒到混合物, 孵育在37° c 为60分钟与晃动在 250 rpm, 传播100µL 到性肉汤 (LB) 琼脂板与相应的抗生素, 在37° c 的夜间孵育, 以 250 rpm 摇动。</li>
	</ol>
</li>
	<li>选择4-6 个单一的殖民地到6毫升新鲜 LB 培养基与相应的抗生素, 并孵化在37° c 过夜与晃动 250 rpm。通过质粒纯化试剂盒从每一夜培养物中提取粒细胞, 按照制造商手册, 然后根据服务提供商的协议发送基因组进行 DNA 测序, 以确认停止密码子在正确位置的突变。</li>
	<li>通过混合1毫升过夜培养和300µL 100% 亚砜, 在-80 ° c 下将应变存储在正确的顺序上。</li>
</ol>2. 乙酰蛋白的表达
<ol><li>插入优化的 acetyllysyl-tRNA 合成酶33的基因, 并优化 tRNAPyl 32到pTech质粒。将 tRNA 合成酶基因置于本构lpp启动子下。将 tRNA 基因置于本构普罗克启动子34下。<ol>
<li>将含有苹果酸脱氢酶的突变标记基因的表达载体34和包含优化的 acetyllysine 合并系统的质粒, 转化为25µL 解冻的合格的大肠杆菌 BL21 (DE3) 细胞热休克在42° c 为十年代和地方在冰为另外5分钟。</li>
		<li>吸管600µL 室温 SOC 介质进入混合物, 孵育在37° c 为60分钟, 晃动在 275 rpm, 传播100µL 到一个板块与100µg/毫升链霉素和50µg/毫升氯霉素, 和孵化过夜在37° c 与晃动在 275 rpm。</li>
	</ol>
</li>
	<li>拿起一个单一的殖民地从板块, 并接种到15毫升新鲜 LB 培养基与100µg/毫升链霉素和50µg/毫升氯霉素在50毫升管夜间在37° c 与晃动的速度为 250 rpm。在1升烧瓶中用抗生素将15毫升过夜培养到300毫升新鲜 LB 培养基, 在37° c 下孵育, 在 250 rpm 时晃动。</li>
	<li>用清水溶解 acetyllysine, 使100毫米库存溶液, 贮存在4° c。添加5毫米 acetyllysine 和20毫米烟酰胺 (抑制剂乙酰) 的增长媒体时, 吸光度达到 0.5, 在 600 nm。<ol>
<li>在37° c 下增加1小时的细胞, 摇动 250 rpm, 然后增加0.5 毫米 IPTG 蛋白表达, 并在25° c 的夜间生长细胞, 以 180 rpm 摇动。 注意:表达条件可能需要对不同的蛋白质进行优化。</li>
	</ol>
</li>
	<li>收集细胞的离心在 3000 x g 在4° c 的15分钟, 丢弃上清液, 用磷酸盐缓冲的生理盐水 (PBS) 缓冲洗涤细胞小球 (10 毫米 Na2HPO4, 1.8 mm,2PO4, 137 毫米氯化钠, 2.7 mm 氯化钾)。收集洗涤的细胞在 1万 x g 在4° c 为5分钟, 放弃上清, 并且存放细胞小球在-80 ° c。</li>
</ol>3. 乙酰蛋白的纯化
<ol><li>解冻冰冻细胞在冰上的颗粒并重新悬挂15毫升的裂解缓冲液 (50 毫米三 (羟甲基) 氨基 (三) pH 7.8, 300 毫米氯化钠, 20 毫米咪唑, 20 毫米烟酰胺), 5 µL β-基和1µL Benzonase 核酸 (250 单位)。</li>
	<li>打破细胞由40赫超声在70% 功率输出与10周期的十年代短脉冲, 其次是三十年代的时间间隔, 冷却形成原油提取物。离心机粗提取物在 2万 x g 为25分钟在4° c。用0.45 µm 膜过滤器过滤上清液, 并将1毫升的镍三酸 (镍-NTA) 树脂平衡和20毫升的水和20毫升的裂解缓冲物载入一列。 注意:如果超声不可用, 也可以用温和的洗涤剂打破细胞。</li>
	<li>用20毫升洗涤缓冲液 (50 mm ph 7.8, 300 毫米氯化钠, 50 毫米咪唑和20毫米烟酰胺) 冲洗柱, 然后洗与2毫升洗脱缓冲液 (50 毫米三 ph 7.8, 300 毫米氯化钠, 150 毫米咪唑, 和20毫米烟酰胺)。</li>
	<li>淡化的洗脱馏分与脱盐缓冲 (25 毫米三 pH 7.8 和10毫米氯化钠) 的 PD-10 柱, 按照制造商的手册。按照布拉德福德蛋白测定试剂的指示, 测定洗蛋白的浓度。盐水蛋白为进一步的实验做好了准备。 注意:使50% 的甘油库存的蛋白质, 并保持在-80 ° c 存储。</li>
</ol>4. 乙酰蛋白的生化特性
<ol><li>
SDS-页和质谱分析。
	<ol>
<li>变性蛋白质与十二烷基硫酸钠 (sds) 样品缓冲 (5 µL 蛋白质样品与2µL 4X sds 样品缓冲) 在2毫升管子在105° c 为 5 min, 离心混合物在 2000 x g 为十年代, 装载到4-20% 钠十二烷基硫酸盐聚丙烯酰胺凝胶 electrophoresis (SDS 页) 凝胶, 并运行在200伏30分钟。</li>
		<li>用蒸馏水清洗凝胶, 轻轻摇动5分钟, 重复过程3次。将水丢弃, 用马斯亮的蓝色污渍将凝胶沾上1小时, 并轻轻摇动。用蒸馏水把凝胶弄脏, 轻轻摇动30分钟, 重复这个 de 污点3次。</li>
		<li>削减 33 kDa 在马斯亮蓝染色的 SDS 页凝胶带, 并发送到质谱仪设施或公司, 以确认 acetyllysine 被纳入设计的立场。 注: 质谱分析的协议在上一次实验之前34。</li>
	</ol>
</li>
	<li>
西部印迹
	<ol>
<li>在步骤4.1 中使用相同的协议运行 SDS 页凝胶。在凝胶奔跑以后, 浸泡胶凝体与转移缓冲 (25 毫米三, 192 毫米甘氨酸, pH 8.3 和20% 甲醇) 为 15 min。</li>
		<li>激活一个0.2 µm, 7 厘米 x 8.5 厘米的聚偏氟 (PVDF) 膜与甲醇为1分钟, 并在准备转移三明治前用转移缓冲液冲洗。 注意:甲醇是危险的情况下, 皮肤接触, 眼睛接触, 摄入, 或吸入。严重过度暴露会导致死亡。</li>
		<li>使从阴极到阳极 (海绵, 滤纸, SDS 页凝胶, PVDF 膜, 滤纸, 海绵) 的转移三明治。将栈放入转印罐中, 在350毫安的恒定电流下运行45分钟。 注意:传输时间可能需要优化。</li>
		<li>用25毫升三缓冲盐水冲洗 PVDF 膜, 0.1% 吐温 20 (TBST) (137 毫米氯化钠, 20 毫米三, 0.1% Tween-20, pH 7.6) 缓冲液为5分钟, 轻柔摇动。在室温下, 用5% 牛血清白蛋白 (BSA) 在 TBST 缓冲膜中阻挡1小时。</li>
		<li>用 HRP 结合的 acetyllysine 抗体孵育膜, 最终浓度为1µg/毫升稀释, 在 TBST ° c 的情况下, 一夜之间用 5% BSA 进行温和摇动。 注意:为了更快的结果, 这一步可以在室温下执行1小时。抗体的稀释可能需要优化。</li>
		<li>用20毫升 TBST 缓冲液冲洗膜, 5 分钟, 轻轻摇动, 重复步骤4次。按照制造商的指示, 将化学发光基板应用到膜上。用电荷耦合器件 (CCD) 相机为基础的成像器捕捉信号。</li>
	</ol>
</li>
</ol>

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作者没有什么可透露的。

非氨基酸 (ncAAs) 的基因合并是基于对一个被分配的密码子的镇压, 主要琥珀色的中止密码子 UAG36,37,38,39, 由 ncAA 充电 tRNA包含相应的子。众所周知, UAG 密码子是公认的释放 factor-1 (RF1) 的细菌, 它也可以抑制附近同源转运从主机的标准氨基酸 (cAAs), 如赖氨酸和酪氨酸40,41。因此, ncaa 在 UAG 密码子中的注册效率取决于 ncaa-转运和 RF1 之间的竞争, 而 ncaa 公司的成立则依赖于 ncaa 的转运和近同源转运之间的竞争。目标乙酰蛋白的低产量和纯度可能是由于引入宿主细胞的正交对的低并网效率造成的。这一问题可以通过增加介质中的 acetyllysine 浓度来解决, 并使用最近优化的 acetyllysine 合并系统, 这增加了 UAG 密码器抑制58次32,33, 这两个acetyllysine 公司的效率和纯度将得到提高。如图 1所示并比较蛋白质的产量, acetyllysine 合并的效率约为 50%, 并且没有可检测的蛋白纯化的细胞窝藏的 ack 注册系统和突变基因的 MDH, 但没有 AcK生长媒介, 表明高纯度 acetyllysine 并网。此外, 质谱分析也没有显示任何 cAAs 的位置140的 MDH, 表明同质性的 acetyllysine 成立。
这种方法有两个主要的限制。首先, 由于 acetyllysine 的 tRNA 与 RF1 的竞争, 以及在上面描述的近同源转运, 目前, 可以同时纳入单个蛋白质的最大 acetyllysine 残留量是三33,42。其次, 细胞有其他类型的乙酰, 抗烟酰胺和可能 deacetylate 某些靶蛋白。因此, 这些蛋白在特定的部位可能达不到100% 的乙酰化。最近, 我们建立了一个 acetyllysine 合并系统, 可作为非 deacetylable 模拟的 acetyllysine43, 因此这个系统可以是一个很好的替代方法在这种情况下。
如前所述, 经典的生化特征赖氨酸乙酰化的方法是使用定点诱变。谷氨酰胺被用作 acetyllysine 的模拟, 精氨酸被用作非乙酰赖氨酸的模拟。但是, 这两种模拟都不是真正的 isosteres, 并且并不总是产生预期的结果30。基因编码扩展策略可以在特定的赖氨酸残留物中生成均匀的乙酰蛋白, 这是乙酰蛋白质的最严格的表征方法。
acetyllysine 的遗传整合系统是从一对 pyrrolysyl-tRNA 合成酶变种, 其同源 tRNA 从Methanosarcinaceae物种, 这也被称为在真核生物44正交。以往的研究表明, 该系统可以应用于哺乳动物细胞和某些动物的蛋白质乙酰化研究39, 因此, 目前的协议可以扩大到哺乳动物细胞, 甚至动物更广泛的应用在医疗研究和工业。此外, 该协议实质上也是用于合并不同类型的 ncAAs 的相同协议, 因此必须对引入到宿主单元中的正交对进行简单的更改。
赖氨酸乙酰 (KDACs) 从蛋白质的乙酰赖氨酸残留物中除去乙酰基,45。sirtuin 型科布是唯一的已知的乙酰在大肠杆菌,可以抑制烟酰胺27。因此, 为了防止在细胞生长和蛋白质纯化过程中产生的乙酰蛋白的脱乙酰度, 应在生长培养基和纯化缓冲液中加入20至50毫米的烟酰胺。一旦纯化, 由于缺乏乙酰, 赖氨酸残留物的乙酰化相对稳定。其次, 为了降低蛋白质中其他赖氨酸残留物的非特异性乙酰化的背景, BL21 (DE3) 菌株被用作表达菌株, 其蛋白乙酰化水平明显低于常用的 K12-derived 菌株46.如图 2所示, 从 BL21 (DE3) 细胞中表达的野生型 MDH 没有被西方印迹所检测到的乙酰化。这是提高靶蛋白乙酰化纯度的另一个重要因素。

转录后的修饰 (PTMs) 的蛋白质发生后, 翻译过程, 并产生了共价键增加功能基团的氨基酸残留, 在几乎所有的生物过程中扮演重要的角色, 包括基因转录, 应激反应, 细胞分化和新陈代谢1,2,3。到目前为止, 已确定了大约400独特的 PTMs4。基因组和蛋白质的复杂性在很大程度上被蛋白质 PTMs, 因为它们调节蛋白质活性和定位, 并影响与其他分子的相互作用, 如蛋白质、核酸、血脂和因子5。
在过去二十年中, 蛋白质乙酰化一直处于 PTMs 研究的前沿6、7、8、9、10、11、12。在组蛋白中首次发现的赖氨酸乙酰化超过50年前的13,14, 已被仔细检查, 并已知存在于超过80转录因子、调节器和各种蛋白质15中, 16,17。对蛋白质乙酰化的研究不仅为我们提供了对其调节机制的更深的了解, 而且也为一些因功能失调而引起的疾病进行了指导治疗18,19,20,21,22,23. 据认为, 赖氨酸乙酰化只发生在真核生物, 但最近的研究表明, 蛋白质乙酰化也发挥关键作用的细菌生理学, 包括趋化性, 耐酸, 活化, 稳定致病性岛屿及其他毒力相关蛋白24,25,26,27,28,29。
一种常用的方法来生化的特点赖氨酸乙酰化是使用定点诱变。谷氨酰胺被用作 acetyllysine 的模拟, 因为它的大小和极性相似。精氨酸被用作非乙酰赖氨酸的模拟, 因为它在生理条件下保留了正电荷, 但不能乙酰。但是, 这两种模拟都不是真正的 isosteres, 并且并不总能产生预期的结果30。最严谨的方法是在特定的赖氨酸残留物中生成均匀的乙酰蛋白, 这对于大多数经典方法来说是困难或不可能的, 因为在自然界中, 赖氨酸乙酰化的低化学计量性7,11。这一挑战已被解开的遗传代码扩展战略, 采用了工程 pyrrolysyl-tRNA 合成酶变种从Methanosarcinaceae物种的收费 tRNAPyl与 acetyllysine, 利用主机用于抑制 mRNA 中的 UAG 停止密码子的平移机械, 并指示在目标蛋白31的设计位置中合并 acetyllysine。最近, 我们优化了这个系统, 改进了 EF Tu 绑定 tRNA32和升级后的 acetyllysyl-tRNA 合成酶33。此外, 我们在苹果酸脱氢酶34和氨-tRNA 合成酶35的乙酰化研究中应用了这种增强的并网系统。本文以苹果酸脱氢酶 (MDH) 为例, 研究了从分子克隆到生物化学鉴定的纯乙酰蛋白的生成方法, 并以此作为演示的例证。

<table border="0" cellpadding="0" cellspacing="0" width="615">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Bradford protein assay</td>
			<td style="width:113px;">Bio-Rad</td>
			<td style="width:119px;">5000006</td>
			<td style="width:167px;">Protein concentration</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">4x Laemmli Sample Buffer</td>
			<td style="width:113px;">Bio-Rad</td>
			<td style="width:119px;">1610747</td>
			<td style="width:167px;">SDS sample buffer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Coomassie G-250 Stain</td>
			<td style="width:113px;">Bio-Rad</td>
			<td style="width:119px;">1610786</td>
			<td style="width:167px;">SDS-PAGE gel staining</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">4-20% SDS-PAGE ready gel</td>
			<td style="width:113px;">Bio-Rad</td>
			<td style="width:119px;">4561093</td>
			<td style="width:167px;">Protein determination</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Ac-K-100 (HRP Conjugate)</td>
			<td style="width:113px;">Cell Signaling</td>
			<td style="width:119px;">6952</td>
			<td style="width:167px;">Antibody</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">IPTG</td>
			<td style="width:113px;">CHEM-IMPEX</td>
			<td style="width:119px;">194</td>
			<td style="width:167px;">Expression inducer</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:216px;">N&#949;-Acetyl-L-lysine</td>
			<td style="width:113px;">CHEM-IMPEX</td>
			<td style="width:119px;">5364</td>
			<td style="width:167px;">Noncanonical amino acid</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">PD-10 desalting column</td>
			<td style="width:113px;">GE Healthcare</td>
			<td style="width:119px;">17085101</td>
			<td style="width:167px;">Desalting</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Q5 Site-Directed Mutagenesis Kit</td>
			<td style="width:113px;">NEB</td>
			<td style="width:119px;">E0554</td>
			<td style="width:167px;">Introducing the stop codon</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">BL21 (DE3) cells</td>
			<td style="width:113px;">NEB</td>
			<td style="width:119px;">C2527</td>
			<td style="width:167px;">Expressing strain</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">QIAprep Spin Miniprep Kit</td>
			<td style="width:113px;">QIAGEN</td>
			<td style="width:119px;">27106</td>
			<td style="width:167px;">Extracting plasmids</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Ni-NTA resin</td>
			<td style="width:113px;">QIAGEN</td>
			<td style="width:119px;">30210</td>
			<td style="width:167px;">Affinity purification resin</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">nicotinamide</td>
			<td style="width:113px;">Sigma-Aldrich</td>
			<td style="width:119px;">N3376</td>
			<td style="width:167px;">Deacetylase inhibitor</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">&#946;-Mercaptoethanol</td>
			<td style="width:113px;">Sigma-Aldrich</td>
			<td style="width:119px;">M6250</td>
			<td style="width:167px;">Reducing agent</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">BugBuster Protein Extraction Reagent</td>
			<td style="width:113px;">Sigma-Aldrich</td>
			<td style="width:119px;">70584</td>
			<td style="width:167px;">Breaking cells</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Benzonase nuclease</td>
			<td style="width:113px;">Sigma-Aldrich</td>
			<td style="width:119px;">E1014</td>
			<td style="width:167px;">DNase</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">ECL Western Blotting Substrate</td>
			<td style="width:113px;">ThermoFisher</td>
			<td style="width:119px;">32106</td>
			<td style="width:167px;">Chemiluminescence</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Premixed LB Broth</td>
			<td style="width:113px;">VWR</td>
			<td style="width:119px;">97064</td>
			<td style="width:167px;">Cell growth medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Bovine serum albumin</td>
			<td style="width:113px;">VWR</td>
			<td style="width:119px;">97061-416</td>
			<td style="width:167px;">western blots blocking</td>
		</tr>
	</tbody>
</table>

a facile protocol to generate site-specifically acetylated proteins in escherichia coli

在蛋白质的特定位置发生的后平移修饰被证明在多种细胞过程中发挥重要作用。其中, 可逆赖氨酸乙酰化是最广泛分布在生活的所有领域之一。尽管已经进行了大量的质谱 acetylome 研究, 但对这些假定的乙酰化靶点的进一步定性是有限的。一个可能的原因是, 它是很难产生纯乙酰蛋白质在理想的位置, 大多数经典的生物化学方法。为了克服这一挑战, 基因编码扩展技术已被应用于使用一对工程 pyrrolysyl tRNA 合成酶变种, 其同源 tRNA 从Methanosarcinaceae物种, 以指导 cotranslational 公司成立acetyllysine 在特定的地点的蛋白质的利益。在首次应用于组蛋白乙酰化的研究中, 这种方法促进了对各种蛋白质的乙酰化研究。在这项工作中, 我们展示了一个简便的协议, 以生产现场特定的乙酰蛋白, 使用模型细菌大肠埃希菌作为宿主。以苹果酸脱氢酶为例。

乙酰 MDH 蛋白的产量为每 1 l 培养15毫克, 而野生型 MDH 为31毫克, 每 1 l 培养。用 SDS-页分析纯化蛋白, 如图 1所示。使用野生类型的 MDH 作为正控制34。从细胞中纯化的 acetyllysine (AcK) 合并系统和突变的mdh基因, 但在生长培养基中没有 AcK, 被用作阴性对照。用 acetyllysine 抗体检测纯化蛋白的赖氨酸乙酰化, 如图 2所示。用串联质谱分析法证实了赖氨酸渣140在苹果酸脱氢酶中的乙酰化, 如图 3所示。
MDH 蛋白的蛋白质序列 (串联 MS 分析的片段是黑体):
MKVAVLGAAGGIGQALALLLKTQLPSGSELSLYDIAPVTPGVAVDLSHIPTAVKIKGFSGEDAT PALEGADVVLISAGVARKPGMDRSDLFNVNAGIVKNLVQQVAKTCPKACIGIITNPVNTTVAIAA EVLKKAGVYDKNKLFGVTTLDIIRSNTFVAELKGKQPGEVEVPVIGGHSGVTILPLLSQVPGV SFTEQEVADLTKRIQNAGTEVVEAKAGGGSATLSMGQAAARFGLSLVRALQGEQGVVECAY VEGDGQYARFFSQPLLLGKNGVEERKSIGTLSAFEQNALEGMLDTLKKDIALGEEFVNK
优化 acetyllysyl tRNA 合成酶的蛋白质序列33:
MDKKPLDVLISATGLWMSRTGTLHKIKHYEISRSKIYIEMACGDHLVVNNSRSCRPARAFRYHKY RKTCKRCRVSDEDINNFLTRSTEGKTSVKVKVVSEPKVKKAMPKSVSRAPKPLENPVSAKAST DTSRSVPSPAKSTPNSPVPTSASAPALTKSQTDRLEVLLNPKDEISLNSGKPFRELESELLSRR KKDLQQIYAEERENYLGKLEREITRFFVDRGFLEIKSPILIPLEYIERMGIDNDTELSKQIFRVDKN FCLRPMMAPNLLNYARKLDRALPDPIKIFEIGPCYRKESDGKEHLEEFTMLNFFQMGSGCTRE NLESIITDFLNHLGIDFKIVGDSCMVYGDTLDVMHGDLELSSAVVGPIPLDREWGIDKPWIGAGF GLERLLKVKHDFKNIKRAARSESYYNGISTNL
优化 tRNA 的基因序列Pyl 32:
GGAAACGTGATCATGTAGATCGAATGGACTCTAAATCCGTTCAGTGGGGTTAGATTCCCC ACGTTTCCGCCA
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57061/57061fig1.jpg"> 图 1: 马斯亮蓝染色 SDS-页凝胶的纯化全长 MDH 及其含 AcK 变种.同样体积的洗脱分数被加载到 SDS 页凝胶。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57061/57061fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57061/57061fig2.jpg"> 图 2: 纯化野生型 MDH 及其含 AcK 变种的印迹.同样体积的洗脱馏分被加载。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57061/57061fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57061/57061fig3.jpg"> 图 3: 含 AcK 的 MDH 变体的 LC/ms 分析.肽的串联质谱 (残滓 135-142) AGVYDKACNK 从纯化乙酰 MDH 变种。KAC表示 AcK 合并。含有 AcK 的肽的部分序列可以从注释的 b 或 y 离子系列中读取。匹配的峰值为红色。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57061/57061fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about A Facile Protocol to Generate Site-Specifically Acetylated Proteins in Escherichia Coli at JoVE.com

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

基因编码扩展是研究广泛的生物过程, 包括蛋白质乙酰化的有力工具。在这里, 我们演示了一个轻便的协议, 利用这种技术, 在特定的地点, 在大肠杆菌细胞生成均匀乙酰蛋白。

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. ...

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JoVE Journal

Immunology and Infection

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

6.8K 次观看.  University of Arkansas. 基因编码扩展是研究广泛的生物过程, 包括蛋白质乙酰化的有力工具。在这里, 我们演示了一个轻便的协议, 利用这种技术, 在特定的地点, 在大肠杆菌细胞生成均匀乙酰蛋白。

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

Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells

The obligate intracellular bacterium Chlamydia elicits a great burden on global public health. C.&#160;trachomatis is the leading bacterial cause of sexually transmitted infection and also the primary cause of preventable blindness in the world. An essential determinant for successful infection of host cells by Chlamydia is the bacterium's ability to manipulate host cell signaling from within a novel, vacuolar compartment called the inclusion. From within the inclusion, Chlamydia acquire nutrients required for their 2-3 day developmental growth, and they additionally secrete a panel of effector proteins onto the cytosolic face of the vacuole membrane and into the host cytosol. Gaps in our understanding of Chlamydia biology, however, present significant challenges for visualizing and analyzing this intracellular compartment. Recently, a reverse-imaging strategy for visualizing the inclusion using GFP expressing host cells was described. This approach rationally exploits the intrinsic impermeability of the inclusion membrane to large molecules such as GFP. In this work, we describe how GFP- or mCherry-expressing host cells are generated for subsequent visualization of chlamydial inclusions. Furthermore, this method is shown to effectively substitute for costly antibody-based enumeration methods, can be used in tandem with other fluorescent labels, such as GFP-expressing Chlamydia, and can be exploited to derive key quantitative data about inclusion membrane growth from a range of Chlamydia species and strains.

Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells

A live cell fluorescent protein based method for illuminating cellular vacuoles (inclusions) containing Chlamydia is described. This strategy enables rapid, automated determination of Chlamydia infectivity in samples and can be used to quantitatively investigate inclusion growth dynamics.

利用荧光蛋白可视化和定量<em&gt;衣原体</em&gt;液泡生长动态的活细胞

The obligate intracellular bacterium Chlamydia elicits a great burden on global public health. C.&#160;trachomatis is the leading bacterial cause of ...

科研

免疫与感染

A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium

In the last decade, C. elegans has emerged as an invertebrate organism to study interactions between hosts and pathogens, including the host defense against gram-negative bacterium Salmonella typhimurium. Salmonella establishes persistent infection in the intestine of C. elegans and results in early death of infected animals. A number of immunity mechanisms have been identified in C. elegans to defend against Salmonella infections. Autophagy, an evolutionarily conserved lysosomal degradation pathway, has been shown to limit the Salmonella replication in C. elegans and in mammals. Here, a protocol is described to infect C. elegans with Salmonella typhimurium, in which the worms are exposed to Salmonella for a limited time, similar to Salmonella infection in humans. Salmonella infection significantly shortens the lifespan of C. elegans. Using the essential autophagy gene bec-1 as an example, we combined this infection method with C. elegans RNAi feeding approach and showed this protocol can be used to examine the function of C. elegans host genes in defense against Salmonella infection. Since C. elegans whole genome RNAi libraries are available, this protocol makes it possible to comprehensively screen for C. elegans genes that protect against Salmonella and other intestinal pathogens using genome-wide RNAi libraries.

A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium

C. elegans has emerged as a new genetic model to study host-pathogen interactions. Here we describe a protocol to infect C. elegans with Salmonella typhimurium coupled with the double-strand RNAi interference technique to examine the role of host genes in defense against Salmonella infection.

一个协议感染<em&gt;秀丽隐杆线虫</em&gt;与<em&gt;鼠伤寒沙门氏菌</em

In the last decade, C. elegans has emerged as an invertebrate organism to study interactions between hosts and pathogens, including the host defense ...

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal features of the natural infection. This protocol describes procedures for the establishment and evaluation of systemic infection due to neuropathogenic Escherichia coli K1 in the neonatal rat. Colonization of the gastrointestinal tract leads to dissemination of the pathogen along the gut-lymph-blood-brain course of infection and the model displays strong age dependency. A strain of E. coli O18:K1 with enhanced virulence for the neonatal rat produces exceptionally high rates of colonization, translocation to the blood compartment and invasion of the meninges following transit through the choroid plexus. As in the human host, penetration of the central nervous system is accompanied by local inflammation and an invariably lethal outcome. The model is of proven utility for studies of the mechanism of pathogenesis, for evaluation of therapeutic interventions and for assessment of bacterial virulence.

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Here, a procedure is described for the establishment of systemic infection in the neonatal rat with cultures of Escherichia coli K1. This non-invasive procedure permits colonization of the gastrointestinal tract, translocation of the pathogen to the systemic circulation, and invasion of the central nervous system at the choroid plexus.

Neuropathogenic的非侵入型<em&gt;大肠杆菌</em&gt;感染乳鼠

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal ...

Phage Phenomics: Physiological Approaches to Characterize Novel Viral Proteins

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage sequences share no homology to current annotated proteins. As a result, a large proportion of phage genes are annotated as hypothetical. This reality heavily affects the annotation of both structural and auxiliary metabolic genes. Here we present phenomic methods designed to capture the physiological response(s) of a selected host during expression of one of these unknown phage genes. Multi-phenotype Assay Plates (MAPs) are used to monitor the diversity of host substrate utilization and subsequent biomass formation, while metabolomics provides bi-product analysis by monitoring metabolite abundance and diversity. Both tools are used simultaneously to provide a phenotypic profile associated with expression of a single putative phage open reading frame (ORF). Representative results for both methods are compared, highlighting the phenotypic profile differences of a host carrying either putative structural or metabolic phage genes. In addition, the visualization techniques and high throughput computational pipelines that facilitated experimental analysis are presented.

Here, we present phenomic approaches for the functional characterization of putative phage genes. Techniques include a developed assay capable of monitoring host anabolic metabolism, the Multi-phenotype Assay Plates (MAPs), in addition to the established method of metabolomics, capable of measuring effects to catabolic metabolism.

噬菌体型组学:生理途径来表征新型病毒蛋白

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage ...

One-step Negative Chromatographic Purification of Helicobacter pylori Neutrophil-activating Protein Overexpressed in Escherichia coli in Batch Mode

Helicobacter pylori neutrophil-activating protein (HP-NAP) is a major virulence factor of Helicobacter pylori (H. pylori). It plays a critical role in H. pylori-induced gastric inflammation by activating several innate leukocytes including neutrophils, monocytes, and mast cells. The immunogenic and immunomodulatory properties of HP-NAP make it a potential diagnostic and vaccine candidate for H. pylori and a new drug candidate for cancer therapy. In order to obtain substantial quantities of purified HP-NAP used for its clinical applications, an efficient method to purify this protein with high yield and purity needs to be established.
In this protocol, we have described a method for one-step negative chromatographic purification of recombinant HP-NAP overexpressed in Escherichia coli (E. coli) by using diethylaminoethyl (DEAE) ion-exchange resins (e.g., Sephadex) in batch mode. Recombinant HP-NAP constitutes nearly 70% of the total protein in E. coli and is almost fully recovered in the soluble fraction upon cell lysis at pH 9.0. Under the optimal condition at pH 8.0, the majority of HP-NAP is recovered in the unbound fraction while the endogenous proteins from E. coli are efficiently removed by the resin.
This purification method using negative mode batch chromatography with DEAE ion-exchange resins yields functional HP-NAP from E. coli in its native form with high yield and purity. The purified HP-NAP could be further utilized for the prevention, treatment, and prognosis of H. pylori-associated diseases as well as cancer therapy.

One-step Negative Chromatographic Purification of Helicobacter pylori Neutrophil-activating Protein Overexpressed in Escherichia coli in Batch Mode

A high yield method for one-step negative purification of recombinant Helicobacter pylori neutrophil-activating protein (HP-NAP) overexpressed in Escherichia coli by using diethylaminoethyl resins in batch mode is described. HP-NAP purified by this method is beneficial for the development of vaccines, drugs, or diagnostics for H. pylori-associated diseases.

一步法负色谱纯化<em&gt;幽门螺杆菌</em&gt;中性粒细胞激活蛋白过度表达<em&gt;大肠杆菌</em&gt;在批处理模式

Helicobacter pylori neutrophil-activating protein (HP-NAP) is a major virulence factor of Helicobacter pylori (H. pylori). It plays a critical role in H. ...

A Protocol to Characterize the Morphological Changes of Clostridium difficile in Response to Antibiotic Treatment

Assessment of antibiotic action with new drug development directed towards anaerobic bacteria is difficult and technically demanding. To gain insight into possible MOA, morphologic changes associated with antibiotic exposure can be visualized using scanning electron microscopy (SEM). Integrating SEM imaging with traditional kill curves may improve our insight into drug action and advance the drug development process. To test this premise, kill curves and SEM studies were conducted using drugs with known but different MOA (vancomycin and metronidazole). C. difficile cells (R20291) were grown with or without the presence of antibiotic for up to 48 h. Throughout the 48 h interval, cells were collected at multiple time points to determine antibiotic efficacy and for imaging on the SEM. Consistent with previous reports, vancomycin and metronidazole had significant bactericidal activity following 24 h of treatment as measured by colony-forming unit (CFU) counting. Using SEM imaging we determined that metronidazole had significant effects on cell length (&#62; 50% reduction in cell length for each antibiotic; P&#60; 0.05) compared to controls and vancomycin. While the phenotypic response to drug treatment has not been documented previously in this manner, they are consistent with the drug&#39;s MOA demonstrating the versatility and reliability of the imaging and measurements and the application of this technique for other experimental compounds.

A Protocol to Characterize the Morphological Changes of Clostridium difficile in Response to Antibiotic Treatment

Antibiotic efficacy is most commonly determined by conducting killing kinetic studies and measuring colony forming units (CFUs). By integrating scanning electron microscopy (SEM) with these standard methods, we can distinguish the pharmacological effects of treatment between different antibiotics.

表征形态变化的议定书<em&gt;艰难梭菌</em&gt;应对抗生素治疗

Assessment of antibiotic action with new drug development directed towards anaerobic bacteria is difficult and technically demanding. To gain insight into ...

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome (HUS), colonize to the intestinal tract of humans. To study the detailed mechanism of EHEC colonization in vivo, it is essential to have animal models to monitor and quantify EHEC colonization. We demonstrate here a mouse-EHEC colonization model by transforming the bioluminescent expressing plasmid to EHEC to monitor and quantify EHEC colonization in living hosts. Animals inoculated with bioluminescence-labeled EHEC show intense bioluminescent signals in mice by detection with a non-invasive in vivo imaging system. After 1 and 2 days post infection, bioluminescent signals could still be detected in infected animals, which suggests that EHEC colonize in hosts for at least 2 days. We also demonstrate that these bioluminescent EHEC locate to mouse intestine, specifically in the cecum and colon, from ex vivo images. This mouse-EHEC colonization model may serve as a tool to advance the current knowledge of the EHEC colonization mechanism.

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

A detailed protocol of a mouse model for enterohemorrhagic E. coli (EHEC) colonization by using bioluminescence-labeled bacteria is presented. The detection of these bioluminescent bacteria by a non-invasive in vivo imaging system in live animals can advance our current understanding of EHEC colonization.

非侵入性 体内生物发光系统检测小鼠宿主肠出血性大肠杆菌的定植

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome ...

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below outlines a method for high efficiency transfer between bacterial strains of a plasmid harboring a transposon containing a kanamycin resistance marker. The plasmid-borne transposase is encoded by a variant tnp gene that inserts the transposon into the genome of the recipient strain with very low insertional bias. This method thus allows the creation of large mutant libraries in which transposons have been inserted into unique genomic positions in a recipient strain of either Escherichia coli or Shigella flexneri bacteria. By using bacterial conjugation, as opposed to other methods such as electroporation or chemical transformation, large libraries with hundreds of thousands of unique clones can be created. This yields high-density insertion libraries, with insertions occurring as frequently as every 4-6 base pairs in non-essential genes. This method is superior to other methods as it allows for an inexpensive, easy to use, and high efficiency method for the creation of a dense transposon insertion library. The transposon library can be used in downstream applications such as transposon sequencing (Tn-Seq), to infer genetic interaction networks, or more simply, in mutational (forward genetic) screens.

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Presented here is a simple method for creating a high-density transposon insertion library in Escherichia coli or Shigella flexneri using bacterial conjugation. This protocol allows the creation of a collection of hundreds of thousands of unique mutants in bacteria by the random genomic insertion of a transposon.

使用细菌共轭的 Enterobacterial 菌株 (如大肠杆菌或志贺菌福氏) 创建稠密的转插入库

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions. DHDDS (dehydrodolichyl diphosphate synthase) is a eukaryotic long-chain cis-PT (forming cis double bonds from the condensation reaction) that catalyzes chain elongation of farnesyl diphosphate (FPP, an allylic diphosphate) via multiple condensations with isopentenyl diphosphate (IPP). DHDDS is of biomedical importance, as a non-conservative mutation (K42E) in the enzyme results in retinitis pigmentosa, ultimately leading to blindness. Therefore, the present protocol was developed in order to acquire large quantities of purified DHDDS, suitable for mechanistic studies. Here, the usage of protein fusion, optimized culture conditions and codon-optimization were used to allow the overexpression and purification of functionally active human DHDDS in E. coli. The described protocol is simple, cost-effective and time sparing. The homology of cis-PT among different species suggests that this protocol may be applied for other eukaryotic cis-PT as well, such as those involved in natural rubber synthesis.

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

A simple protocol for overexpression and purification of codon-optimized, human cis-prenyltransferase, under non-denaturing conditions, from Escherichia coli, is described, along with an enzymatic activity assay. This protocol can be generalized for production of other cis- prenyltransferase proteins in quantity and quality suitable for mechanistic studies.

人类的过表达和纯化<em&gt;顺</emβ-肾上腺素转移酶<em&gt;大肠杆菌</em

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

Using Enhanced Green Fluorescence Protein-expressing Escherichia Coli to Assess Mouse Peritoneal Macrophage Phagocytosis

This manuscript describes a simple and reproducible method to perform a phagocytosis assay. The first part of this method involves building a pET-SUMO-EGFP vector (SUMO = small ubiquitin-like modifier) and expressing enhanced green fluorescence protein (EGFP) in Escherichia coli (BL21DE). EGFP-expressing E. coli is coincubated with macrophages for 1 h at 37 &#176;C; the negative control group is incubated on ice for the same amount of time. Then, the macrophages are ready for assessment. The advantages of this technique include its simple and straightforward steps, and phagocytosis can be measured by both flow cytometer and fluorescence microscope. The EGFP-expressing E. coli are stable and display a strong fluorescence signal even after the macrophages are fixed with paraformaldehyde. This method is not only suitable for the assessment of macrophage cell lines or primary macrophages in vitro but also suitable for the evaluation of granulocyte and monocyte phagocytosis in peripheral blood mononuclear cells. The results show that the phagocytic capability of peritoneal macrophages from young (eight-week-old) mice is higher than that of macrophages from aged (16-month-old) mice. In summary, this method measures macrophage phagocytosis and is suitable for studying the innate immune system function.

Using Enhanced Green Fluorescence Protein-expressing Escherichia Coli to Assess Mouse Peritoneal Macrophage Phagocytosis

Here, we present a protocol to assess mouse peritoneal macrophage phagocytosis using enhanced green fluorescence protein-expressing Escherichia coli.