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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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+encoding+N&#949;-acetyllysine+in+recombinant+proteins.">Genetically encoding N&#949;-acetyllysine in recombinant proteins.</a> Nat chem biol. 4 (4), 232-234 (2008).</li><li>Fan, C., Xiong, H., Reynolds, N. M., Soll, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rationally+evolving+tRNAPyl+for+efficient+incorporation+of+noncanonical+amino+acids.">Rationally evolving tRNAPyl for efficient incorporation of noncanonical amino acids.</a> Nucleic Acids Res. 43 (22), e156 (2015).</li><li>Bryson, D., 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=Continuous+directed+evolution+of+aminoacyl-tRNA+synthetases+to+alter+amino+acid+specificity+and+enhance+activity.">Continuous directed evolution of aminoacyl-tRNA synthetases to alter amino acid specificity and enhance activity.</a> Nat Chem Biol. , (2017).</li><li>Venkat, S., Gregory, C., Sturges, J., Gan, Q., Fan, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+the+Lysine+Acetylation+of+Malate+Dehydrogenase.">Studying the Lysine Acetylation of Malate Dehydrogenase.</a> J Mol Biol. 429 (9), 1396-1405 (2017).</li><li>Venkat, S., Gregory, C., Gan, Q., Fan, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+characterization+of+the+lysine+acetylation+of+tyrosyl-tRNA+synthetase+in+Escherichia+coli.">Biochemical characterization of the lysine acetylation of tyrosyl-tRNA synthetase in Escherichia coli.</a> Chembiochem. 18 (19), 1928-1934 (2017).</li><li>Liu, C. C., 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=Adding+new+chemistries+to+the+genetic+code.">Adding new chemistries to the genetic code.</a> Annu Rev Biochem. 79, 413-444 (2010).</li><li>Mukai, T., Lajoie, M. J., Englert, M., Soll, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rewriting+the+Genetic+Code.">Rewriting the Genetic Code.</a> Annu Rev Microbiol. , (2017).</li><li>O'Donoghue, P., Ling, J., Wang, Y. S., Soll, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Upgrading+protein+synthesis+for+synthetic+biology.">Upgrading protein synthesis for synthetic biology.</a> Nat Chem Biol. 9 (10), 594-598 (2013).</li><li>Chin, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expanding+and+reprogramming+the+genetic+code+of+cells+and+animals.">Expanding and reprogramming the genetic code of cells and animals.</a> Annu Rev Biochem. 83, 379-408 (2014).</li><li>O'Donoghue, 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=Near-cognate+suppression+of+amber,+opal+and+quadruplet+codons+competes+with+aminoacyl-tRNAPyl+for+genetic+code+expansion.">Near-cognate suppression of amber, opal and quadruplet codons competes with aminoacyl-tRNAPyl for genetic code expansion.</a> FEBS Lett. 586 (21), 3931-3937 (2012).</li><li>Aerni, H. R., Shifman, M. A., Rogulina, S., O'Donoghue, P., Rinehart, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revealing+the+amino+acid+composition+of+proteins+within+an+expanded+genetic+code.">Revealing the amino acid composition of proteins within an expanded genetic code.</a> Nucleic Acids Res. 43 (2), e8 (2015).</li><li>Huang, Y., 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+convenient+method+for+genetic+incorporation+of+multiple+noncanonical+amino+acids+into+one+protein+in+Escherichia+coli.">A convenient method for genetic incorporation of multiple noncanonical amino acids into one protein in Escherichia coli.</a> Mol Biosyst. 6 (4), 683-686 (2010).</li><li>Venkat, S., 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=Genetically+encoding+thioacetyl-lysine+as+a+nondeacetylatable+analog+of+lysine+acetylation+in+Escherichia+coli.">Genetically encoding thioacetyl-lysine as a nondeacetylatable analog of lysine acetylation in Escherichia coli.</a> FEBS Open. , (2017).</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> Biochim Biophys Acta. 1844 (6), 1059-1070 (2014).</li><li>Gregoretti, I. V., Lee, Y. M., Goodson, H. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+evolution+of+the+histone+deacetylase+family:+functional+implications+of+phylogenetic+analysis.">Molecular evolution of the histone deacetylase family: functional implications of phylogenetic analysis.</a> J Mol Biol. 338 (1), 17-31 (2004).</li><li>Weinert, B. 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=Acetyl-phosphate+is+a+critical+determinant+of+lysine+acetylation+in+E.+coli.">Acetyl-phosphate is a critical determinant of lysine acetylation in E. coli.</a> Mol cell. 51 (2), 265-272 (2013).</li></ol>

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,<sup class="xr...

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

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. ...

a-facile-protocol-to-generate-site-specifically-acetylated-proteins

Universidad de Cartagena UDEC

Research

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

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 ...

科研

生物化学

Deacetylation Assays to Unravel the Interplay between Sirtuins (SIRT2) and Specific Protein-substrates

Acetylation has emerged as an important post-translational modification (PTM) regulating a plethora of cellular processes and functions. This is further supported by recent findings in high-resolution mass spectrometry based proteomics showing that many new proteins and sites within these proteins can be acetylated. However the identity of the enzymes regulating these proteins and sites is often unknown. Among these enzymes, sirtuins, which belong to the class III histone lysine deacetylases, have attracted great interest as enzymes regulating the acetylome under different physiological or pathophysiological conditions. Here we describe methods to link SIRT2, the cytoplasmic sirtuin, with its substrates including both in vitro and in vivo deacetylation assays. These assays can be applied in studies focused on other members of the sirtuin family to unravel the specific role of sirtuins and are necessary in order to establish the regulatory interplay of specific deacetylases with their substrates as a first step to better understand the role of protein acetylation. Furthermore, such assays can be used to distinguish functional acetylation sites on a protein from what may be non-regulatory acetylated lysines, as well as to examine the interplay between a deacetylase and its substrate in a physiological context.

Deacetylation Assays to Unravel the Interplay between Sirtuins SIRT2 and Specific Protein-substrates

This protocol describes the required steps to execute in vitro and in vivo deacetylation assays in order to establish the role of proteins as specific deacetylation substrates for sirtuins and further study the role of reversible - lysine acetylation as a post-translational modification.

脱乙酰测定解开蛋白特异底物乙酰化酶之间的Interplay（SIRT2）和

Acetylation has emerged as an important post-translational modification (PTM) regulating a plethora of cellular processes and functions. This is further ...

化学

Expression, Isolation, and Purification of Soluble and Insoluble Biotinylated Proteins for Nerve Tissue Regeneration

Recombinant protein engineering has utilized Escherichia coli (E. coli) expression systems for nearly 4 decades, and today E. coli is still the most widely used host organism. The flexibility of the system allows for the addition of moieties such as a biotin tag (for streptavidin interactions) and larger functional proteins like green fluorescent protein or cherry red protein. Also, the integration of unnatural amino acids like metal ion chelators, uniquely reactive functional groups, spectroscopic probes, and molecules imparting post-translational modifications has enabled better manipulation of protein properties and functionalities. As a result this technique creates customizable fusion proteins that offer significant utility for various fields of research. More specifically, the biotinylatable protein sequence has been incorporated into many target proteins because of the high affinity interaction between biotin with avidin and streptavidin. This addition has aided in enhancing detection and purification of tagged proteins as well as opening the way for secondary applications such as cell sorting. Thus, biotin-labeled molecules show an increasing and widespread influence in bioindustrial and biomedical fields. For the purpose of our research we have engineered recombinant biotinylated fusion proteins containing nerve growth factor (NGF) and semaphorin3A (Sema3A) functional regions. We have reported previously how these biotinylated fusion proteins, along with other active protein sequences, can be tethered to biomaterials for tissue engineering and regenerative purposes. This protocol outlines the basics of engineering biotinylatable proteins at the milligram scale, utilizing&#160; a T7 lac inducible vector and E. coli expression hosts, starting from transformation to scale-up and purification.

Developing biotinylatable fusion proteins has many potential applications in various fields of research. Recombinant protein engineering is a straight forward procedure that is cost-effective, providing high yields of custom-designed proteins.

表达，分离，纯化可溶性和不溶性蛋白质生物素对神经组织再生

Recombinant protein engineering has utilized Escherichia coli (E. coli) expression systems for nearly 4 decades, and today E. coli is still the most ...

生物工程

Monitoring the Assembly of a Secreted Bacterial Virulence Factor Using Site-specific Crosslinking

This article describes a method to detect and analyze dynamic interactions between a protein of interest and other factors in vivo. Our method is based on the amber suppression technology that was originally developed by Peter Schultz and colleagues1. An amber mutation is first introduced at a specific codon of the gene encoding the protein of interest. The amber mutant is then expressed in E. coli together with genes encoding an amber suppressor tRNA and an amino acyl-tRNA synthetase derived from Methanococcus jannaschii. Using this system, the photo activatable amino acid analog p-benzoylphenylalanine (Bpa) is incorporated at the amber codon. Cells are then irradiated with ultraviolet light to covalently link the Bpa residue to proteins that are located within 3-8 &#197;. Photocrosslinking is performed in combination with pulse-chase labeling and immunoprecipitation of the protein of interest in order to monitor changes in protein-protein interactions that occur over a time scale of seconds to minutes. We optimized the procedure to study the assembly of a bacterial virulence factor that consists of two independent domains, a domain that is integrated into the outer membrane and a domain that is translocated into the extracellular space, but the method can be used to study many different assembly processes and biological pathways in both prokaryotic and eukaryotic cells. In principle interacting factors and even specific residues of interacting factors that bind to a protein of interest can be identified by mass spectrometry.

This article illustrates the use of pulse-chase radio labeling in combination with site-specific photocrosslinking to monitor interactions between a protein of interest and other factors in E. coli. Unlike traditional chemical cross-linking methods, this approach generates high resolution &#8220;snapshots&#8221; of an ordered assembly pathway in a living cell.

监视分泌细菌毒力因子的组装使用站点特定的交联

This article describes a method to detect and analyze dynamic interactions between a protein of interest and other factors in vivo. Our method is based on ...

免疫与感染

Escherichia coli-Based Cell-Free Protein Synthesis: Protocols for a robust, flexible, and accessible platform technology

Over the last 50 years, Cell-Free Protein Synthesis (CFPS) has emerged as a powerful technology to harness the transcriptional and translational capacity of cells within a test tube. By obviating the need to maintain the viability of the cell, and by eliminating the cellular barrier, CFPS has been foundational to emerging applications in biomanufacturing of traditionally challenging proteins, as well as applications in rapid prototyping for metabolic engineering, and functional genomics. Our methods for implementing an E. coli-based CFPS platform allow new users to access many of these applications. Here, we describe methods to prepare extract through the use of enriched media, baffled flasks, and a reproducible method of tunable sonication-based cell lysis. This extract can then be used for protein expression capable of producing 900 &#181;g/mL or more of super folder green fluorescent protein (sfGFP) in just 5 h from experimental setup to data analysis, given that appropriate reagent stocks have been prepared beforehand. The estimated startup cost of obtaining reagents is $4,500 which will sustain thousands of reactions at an estimated cost of $0.021 per &#181;g of protein produced or $0.019 per &#181;L of reaction. Additionally, the protein expression methods mirror the ease of the reaction setup seen in commercially available systems due to optimization of reagent pre-mixes, at a fraction of the cost. In order to enable the user to leverage the flexible nature of the CFPS platform for broad applications, we have identified a variety of aspects of the platform that can be tuned and optimized depending on the resources available and the protein expression outcomes desired.

Escherichia coli-Based Cell-Free Protein Synthesis: Protocols for a robust, flexible, and accessible platform technology

This protocol details the steps, costs, and equipment necessary to generate E. coli-based cell extracts and implement in vitro protein synthesis reactions within 4 days or less. To leverage the flexible nature of this platform for broad applications, we discuss reaction conditions that can be adapted and optimized.

大肠杆菌无细胞蛋白合成: 一种强大、灵活和可访问的平台技术的协议

Over the last 50 years, Cell-Free Protein Synthesis (CFPS) has emerged as a powerful technology to harness the transcriptional and translational capacity ...

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

Acetylated histone proteins can be easily expressed in Escherichia coli encoding a mutant, N&#949;-acetyl-lysine (AcK)-specific Methanosarcina mazi pyrrolysine tRNA-synthetase (MmAcKRS1) and its cognate tRNA (tRNAPyl) to assemble reconstituted mononucleosomes with site specific acetylated histones. MmAcKRS1 and tRNAPyl deliver AcK at an amber mutation site in the mRNA of choice during translation in Escherichia coli. This technique has been used extensively to incorporate AcK at H3 lysine sites. Pyrrolysyl-tRNA synthetase (PylRS) can also be easily evolved to incorporate other noncanonical amino acids (ncAAs) for site specific protein modification or functionalization. Here we detail a method to incorporate AcK using the MmAcKRS1 system into histone H3 and integrate acetylated H3 proteins into reconstituted mononucleosomes. Acetylated reconstituted mononucleosomes can be used in biochemical and binding assays, structure determination, and more. Obtaining modified mononucleosomes is crucial for designing experiments related to discovering new interactions and understanding epigenetics.

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

Here we present a method to express acetylated histone proteins using genetic code expansion and assemble reconstituted nucleosomes in vitro.

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 ...

Engineering 'Golden' Fluorescence by Selective Pressure Incorporation of Non-canonical Amino Acids and Protein Analysis by Mass Spectrometry and Fluorescence

Fluorescent proteins are fundamental tools for the life sciences, in particular for fluorescence microscopy of living cells. While wild-type and engineered variants of the green fluorescent protein from Aequorea victoria (avGFP) as well as homologs from other species already cover large parts of the optical spectrum, a spectral gap remains in the near-infrared region, for which avGFP-based fluorophores are not available. Red-shifted fluorescent protein (FP) variants would substantially expand the toolkit for spectral unmixing of multiple molecular species, but the naturally occurring red-shifted FPs derived from corals or sea anemones have lower fluorescence quantum yield and inferior photo-stability compared to the avGFP variants. Further manipulation and possible expansion of the chromophore&#39;s conjugated system towards the far-red spectral region is also limited by the repertoire of 20 canonical amino acids prescribed by the genetic code. To overcome these limitations, synthetic biology can achieve further spectral red-shifting via insertion of non-canonical amino acids into the chromophore triad. We describe the application of SPI to engineer avGFP variants with novel spectral properties. Protein expression is performed in a tryptophan-auxotrophic E. coli strain and by supplementing growth media with suitable indole precursors. Inside the cells, these precursors are converted to the corresponding tryptophan analogs and incorporated into proteins by the ribosomal machinery in response to UGG codons. The replacement of Trp-66 in the enhanced &#34;cyan&#34; variant of avGFP (ECFP) by an electron-donating 4-aminotryptophan results in GdFP featuring a 108 nm Stokes shift and a strongly red-shifted emission maximum (574 nm), while being thermodynamically more stable than its predecessor ECFP. Residue-specific incorporation of the non-canonical amino acid is analyzed by mass spectrometry. The spectroscopic properties of GdFP are characterized by time-resolved fluorescence spectroscopy as one of the valuable applications of genetically encoded FPs in life sciences.

Synthetic biology enables the engineering of proteins with unprecedented properties using the co-translational insertion of non-canonical amino acids. Here, we presented how a spectrally red-shifted variant of a GFP-type fluorophore with novel fluorescence spectroscopic properties, termed &#34;gold&#34; fluorescent protein (GdFP), is produced in E. coli via selective pressure incorporation (SPI).

用质谱和荧光法研究非规范氨基酸和蛋白质分析的选择性压力下工程 "金" 荧光

Fluorescent proteins are fundamental tools for the life sciences, in particular for fluorescence microscopy of living cells. While wild-type and ...

Peptide Scanning-assisted Identification of a Monoclonal Antibody-recognized Linear B-cell Epitope

The identification of an antigenic epitope by the immune system allows for the understanding of the protective mechanism of neutralizing antibodies that may facilitate the development of vaccines and peptide drugs. Peptide scanning is a simple and efficient method that straightforwardly maps the linear epitope recognized by a monoclonal antibody (mAb). Here, the authors present an epitope determination methodology involving serially truncated recombinant proteins, synthetic peptide design, and dot-blot hybridization for the antigenic recognition of nervous necrosis virus coat protein using a neutralizing mAb. This technique relies on the dot-blot hybridization of synthetic peptides and mAbs on a polyvinylidene fluoride (PVDF) membrane. The minimum antigenic region of a viral coat protein recognized by the RG-M56 mAb can be narrowed down by step-by-step trimmed peptide mapping onto a 6-mer peptide epitope. In addition, alanine scanning mutagenesis and residue substitution can be performed to characterize the binding significance of each amino acid residue making up the epitope. The residues flanking the epitope site were found to play critical roles in peptide conformation regulation. The identified epitope peptide may be used to form crystals of epitope peptide-antibody complexes for an x-ray diffraction study and functional competition, or for therapeutics.

Here, the authors present a simple and efficient protocol to define a linear antigenic epitope using a purified monoclonal antibody and peptide scanning through dot-blot hybridization. The identified epitope can then be used in therapeutic and diagnostic applications.

肽扫描辅助一个单克隆抗体识别的线性B细胞表位的鉴定

The identification of an antigenic epitope by the immune system allows for the understanding of the protective mechanism of neutralizing antibodies that ...

Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins

Stromal interaction molecule-1 (STIM1) is a type-I transmembrane protein located on the endoplasmic reticulum (ER) and plasma membranes (PM). ER-resident STIM1 regulates the activity of PM Orai1 channels in a process known as store operated calcium (Ca2+) entry which is the principal Ca2+ signaling process that drives the immune response. STIM1 undergoes post-translational N-glycosylation at two luminal Asn sites within the Ca2+ sensing domain of the molecule. However, the biochemical, biophysical, and structure biological effects of N-glycosylated STIM1 were poorly understood until recently due to an inability to readily obtain high levels of homogeneous N-glycosylated protein. Here, we describe the implementation of an in vitro chemical approach which attaches glucose moieties to specific protein sites applicable to understanding the underlying effects of N-glycosylation on protein structure and mechanism. Using solution nuclear magnetic resonance spectroscopy we assess both efficiency of the modification as well as the structural consequences of the glucose attachment with a single sample. This approach can readily be adapted to study the myriad glycosylated proteins found in nature.

Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins

Biochemical and structural analyses of glycosylated proteins require relatively large amounts of homogeneous samples. Here, we present an efficient chemical method for site-specific glycosylation of recombinant proteins purified from bacteria by targeting reactive Cys thiols.

靶向半胱氨酸硫用于体外重组蛋白的特异糖基化

Stromal interaction molecule-1 (STIM1) is a type-I transmembrane protein located on the endoplasmic reticulum (ER) and plasma membranes (PM). ER-resident ...

Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology

Ideal cell-free expression systems can theoretically emulate an in vivo cellular environment in a controlled in vitro platform.1 This is useful for expressing proteins and genetic circuits in a controlled manner as well as for providing a prototyping environment for synthetic biology.2,3 To achieve the latter goal, cell-free expression systems that preserve endogenous Escherichia coli transcription-translation mechanisms are able to more accurately reflect in vivo cellular dynamics than those based on T7 RNA polymerase transcription. We describe the preparation and execution of an efficient endogenous E. coli based transcription-translation (TX-TL) cell-free expression system that can produce equivalent amounts of protein as T7-based systems at a 98% cost reduction to similar commercial systems.4,5 The preparation of buffers and crude cell extract are described, as well as the execution of a three tube TX-TL reaction. The entire protocol takes five days to prepare and yields enough material for up to 3000 single reactions in one preparation. Once prepared, each reaction takes under 8 hr from setup to data collection and analysis. Mechanisms of regulation and transcription exogenous to E. coli, such as lac/tet repressors and T7 RNA polymerase, can be supplemented.6 Endogenous properties, such as mRNA and DNA degradation rates, can also be adjusted.7 The TX-TL cell-free expression system has been demonstrated for large-scale circuit assembly, exploring biological phenomena, and expression of proteins under both T7- and endogenous promoters.6,8 Accompanying mathematical models are available.9,10 The resulting system has unique applications in synthetic biology as a prototyping environment, or "TX-TL biomolecular breadboard."

Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology

This five-day protocol outlines all steps, equipment, and supplemental software necessary for creating and running an efficient endogenous Escherichia coli based TX-TL cell-free expression system from scratch. With reagents, the protocol takes 8 hours or less to setup a reaction, collect, and process data.

协议用于实现<em&gt;大肠杆菌</em&gt;基于TX-TL无细胞表达系统合成生物学

Ideal cell-free expression systems can theoretically emulate an in vivo cellular environment in a controlled in vitro platform.1 This is useful for ...

在大肠杆菌中生成特异的乙酰蛋白的简易协议

摘要

探索更多视频

此视频中的章节

高效抗体标记由应变叠氮推广 - 炔环加成特定站点

脱乙酰测定解开蛋白特异底物乙酰化酶之间的Interplay（SIRT2）和

表达，分离，纯化可溶性和不溶性蛋白质生物素对神经组织再生

监视分泌细菌毒力因子的组装使用站点特定的交联

大肠杆菌无细胞蛋白合成: 一种强大、灵活和可访问的平台技术的协议

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

用质谱和荧光法研究非规范氨基酸和蛋白质分析的选择性压力下工程 "金" 荧光

肽扫描辅助一个单克隆抗体识别的线性B细胞表位的鉴定

靶向半胱氨酸硫用于体外重组蛋白的特异糖基化

协议用于实现

在大肠杆菌中生成特异的乙酰蛋白的简易协议

摘要

探索更多视频

此视频中的章节

高效抗体标记由应变叠氮推广 - 炔环加成特定站点

脱乙酰测定解开蛋白特异底物乙酰化酶之间的Interplay（SIRT2）和

表达，分离，纯化可溶性和不溶性蛋白质生物素对神经组织再生

监视分泌细菌毒力因子的组装使用站点特定的交联

大肠杆菌无细胞蛋白合成: 一种强大、灵活和可访问的平台技术的协议

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

用质谱和荧光法研究非规范氨基酸和蛋白质分析的选择性压力下工程 "金" 荧光

肽扫描辅助一个单克隆抗体识别的线性B细胞表位的鉴定

靶向半胱氨酸硫用于体外重组蛋白的特异糖基化

协议用于实现

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