这项研究得到了全球前沿研究项目 (NRF-2015M3A6A8065833) 的支持, 并通过韩国政府资助的韩国国家研究基金会 (2018R1A6A1A03024940) (NRF) 进行了基础科学研究项目。

1. 质粒结构
<ol><li>构建一种表达质粒 (pBAD-双 TPL-gfp), 在本构启动子和绿色荧光蛋白 (gfp) 基因的控制下表达柠檬酸杆菌 freundii的 TPL 基因, 并在其控制下对其6标记araBAD 启动子。对于 pBAD-dual-TPL-GFP-E90TAG, 使用站点定向的突变协议, 用琥珀密码子 (标记) 替换多巴的站点 (E90) 的密码子。我们在上一份报告18中描述了建造这种质粒的细节。</li>
	<li>构建 tRNA/aaRS 表达质粒 (pEVOL-DHPRS2)18,19用于多巴基因的合并。pEVOL 是一种特殊的质粒载体, 表达了两个 aaRS 基因的独立启动子, 并在前一份报告中描述了质粒的详细信息19。</li>
	<li>使用商业上可用的质粒制备试剂盒获得高纯度质粒 dna。如有必要, 用琼脂糖凝胶电泳检查准备的 dna 纯度。</li>
</ol>2. 文化准备
<ol><li>
穿孔
	<ol>
<li>用电控大肠杆菌DH10β菌株进行实验。将每种质粒 DNA 的1µL (pEVOL-DHPRS2 和 pBAD-dual-TPL-GFP-E90TAG) 添加到20µL 的合格细胞中。用吸管将它们轻轻地混合成均匀的混合物。</li>
		<li>将混合物转移到电穿孔小试管。把试管放在电穿孔室里。将试管推入室内, 使试管室接触牢固。</li>
		<li>冲击试管, 使用25µF 和2.5 伏的0.1 厘米小试管。</li>
		<li>加入1毫升的 prewarmed 超级最佳汤 (SOC), 含有 2% (w/v) 胰蛋白胨, 0.5% (瓦特/v) 酵母提取物, 10 毫米氯化钠, 2.5 毫米氯化钾, 10 毫米氯化镁2, 和20毫米葡萄糖, 到试管, 并转移细胞到培养管。用颤抖的方法在37摄氏度的1小时内孵化, 以拯救细胞。</li>
		<li>在含有35µg/毫升氯霉素和100µg/毫升氨苄西林的溶源性汤 (LB) 琼脂板上传播 10-100 µL 的获救细胞。孵育细胞在37°c 16 小时。</li>
	</ol>
</li>
	<li>
起动机培养制剂
	<ol>
<li>接种5毫升含有抗生素的 LB 培养基中的单个转化的菌落。用颤抖的方法在37摄氏度孵化出12小时的蚁群。</li>
	</ol>
</li>
</ol>3. 生物合成系统对 GFP-E90DOPA 的表达和纯化
<ol><li>
生物合成系统 GFP-E90DOPA 的表达
	<ol>
<li>将起始培养 (2 毫升) 转移到100毫升的 PA-5052 中等20 (50 毫米 Na2HPO4, 50 毫米2PO4, 25 毫米 [NH4]2所以4, 2 毫米 MgSO4, 0.1% [w/v] 痕量金属, 0.5% [瓦特/v] 甘油,0.05% [瓦特/v] 葡萄糖, 和 5% [w/v] 氨基酸 [pH 7.25]) 含有35µg/毫升氯霉素, 100 µg/毫升氨苄西林, 100 毫米丙酮酸, 10 毫米邻苯二酚, 25 毫米氨, 300 µM dithiothreitol (德勤) 其次是孵化在30°c 与震动。</li>
		<li>当光学密度 (OD) 在550毫微米达到0.8 时, 加入2毫升 0.2% (瓦特/v) l-阿拉伯糖。用颤抖的方法在30摄氏度孵化样品12小时。</li>
		<li>旋转 1.1万 x g 的细胞5分钟, 丢弃上清, 并冻结细胞颗粒在-80 摄氏度。</li>
	</ol>
</li>
	<li>
细胞裂解
	<ol>
<li>在冰上解冻细胞颗粒, 并用重悬10毫升的溶解缓冲液中含有50毫米 NaH2PO4 (pH 8.0), 10 毫米咪唑和300毫米氯化钠的细胞。</li>
		<li>油脂实验悬浮细胞在冰上的10分钟使用80% 超声波振幅与脉冲二十五年代和三十五年代。</li>
		<li>在 1.8万 x g 4 摄氏度处旋转细胞裂解液15分钟. 将上清液转移到新鲜的试管中进行提纯并丢弃颗粒。</li>
	</ol>
</li>
	<li>
镍-NTA 亲和层析法
	<ol>
<li>加入镍 NTA (nitrilotriacetic 酸) 树脂 (300 µL 树脂悬浮液100毫升培养) 到上清, 并将蛋白质结合到4摄氏度的镍 NTA 树脂上, 轻轻摇动悬浮液 1 h。</li>
		<li>将悬浮液转移到聚丙烯柱上, 用5毫升的洗涤缓冲液洗涤树脂 3x, 其中含有50毫米 NaH2PO4 (pH 8.0)、20毫米咪唑和300毫米氯化钠。</li>
		<li>洗脱蛋白质3x 与300µL 洗脱缓冲器包含50毫米 NaH2PO4 (pH 8.0), 250 毫米咪唑和300毫米氯化钠。</li>
		<li>通过测量 280 nm 的吸光度测定蛋白质浓度。用蛋白质消光系数计算器 (如https://web.expasy.org/protparam/) 和独立测量的 GFP-E90DOPA 计算了 280 nm 的消光系数 (24630 米-1厘米-1);coefficiennt (2630 米-1厘米-1) 为巴胺。作为一种替代方法, 使用布拉德福德蛋白21测定蛋白质浓度。</li>
	</ol>
</li>
</ol>4. 纯化 GFP-E90DOPA 的齐聚
<ol><li>在含20毫米 Na300GFP-E90DOPA50 (pH 2) 和4毫米氯化钠的磷酸盐缓冲液中, 添加1µL 的高碘酸钠在 H2O (6 毫米) (1.0 equiv) 到 GFP-E90DOPA) 到8.0 µM (300 HPO) 的溶液中。</li>
	<li>允许齐聚反应以25摄氏度为48小时进行。</li>
	<li>用月桂酸钠-聚丙烯酰胺凝胶电泳法分析反应混合物 (SDS 页), 如步骤6所述。</li>
</ol>5. GFP-E90DOPA 与炔烃探针的共轭 SPOCQ
<ol><li>在 H2O (4.0 毫米) (10 ADIBO) 中添加1µL Cy5.5 链接的 azadibenzocyclooctyne (Cy5.5 equiv)22 . GFP-E90DOPA) 到溶液中的20µL 的 GFP-E90DOPA (20 µM) 在磷酸盐缓冲器含有50毫米 Na2HPO4 (pH 8.0) 和300毫米氯化钠。</li>
	<li>将高碘酸钠的1µL 在 H2O (400 µM) (1.0 equiv GFP-E90DOPA) 中加入反应混合物。</li>
	<li>允许 SPOCQ 反应在25摄氏度进行1小时。如果使用光敏感探头, 请用铝箔覆盖反应容器。</li>
</ol>6. 标记的 GFP 的纯化
<ol><li>通过添加磷酸盐缓冲剂稀释 SPOCQ 反应混合物, 含有50毫米 Na2HPO4 (pH 8.0) 和300毫米氯化钠, 最多500µL。</li>
	<li>将稀释后的溶液转移到离心过滤器旋压柱上。离心机的旋转柱在 1.4万 x g在4°c 15 分钟, 并放弃流经。重复此步骤 3x, 以删除任何多余的 Cy5.5-ADIBO。</li>
	<li>将纯化样品从自旋柱转移到1.5 毫升离心管。</li>
	<li>另外, 对同一缓冲液进行透析纯化。</li>
	<li>将纯化的 GFP 保存在4摄氏度。</li>
</ol>7. SDS 页分析和荧光凝胶扫描
<ol><li>通过添加5µL 蛋白质样品缓冲器 (见材料表) 和2µL (1.00 米) 到13µL 的纯化, 或粗, 标记为 GFP (13.6 µM 或0.38 毫克/毫升), 为 SDS 页分析准备蛋白质样品。对于寡聚 gfp 的分析, 使用更高浓度的 gfp (67.8 µM 或1.9 毫克/毫升)。变性蛋白质的孵化, 在95°c 10 分钟。</li>
	<li>将 4%-12% 双三页凝胶盒 (购买的预制凝胶盒) 放置到电泳单元中, 并添加一个正在运行的缓冲器 (见材料表)。加载蛋白质样品和分子量标记。在 200 v 进行电泳 35-40 分钟。 注意: 通过在黑暗中执行所有的过程, 避免对蛋白质样品的任何曝光, 以尽量减少荧光探针的光漂白。</li>
	<li>使用荧光扫描仪进行荧光成像。将蛋白质凝胶从电泳中包裹起来, 放在荧光扫描仪上。使用适当的波长设置扫描凝胶。对于 Cy5.5, 请选择扫描仪软件中提供的 Cy5 模式。</li>
	<li>用商业蛋白染色试剂染色凝胶。</li>
</ol>8. 胰蛋白酶消化 MALDI 分析
<ol><li>孵育100µL 的纯化 GFP-E90DOPA (2.2 毫克/毫升或80µM) 在一个缓冲区中含有50毫米三 HCl (pH 8.0), 0.1% (w/v) SDS, 25 毫米, 在60°c 为 1 h。</li>
	<li>在2O (IAA, 4.0 米) (500 equiv GFP-E90DOPA) 中添加1µL Iodoacetamide 在同一缓冲区中的纯化 GFP-E90DOPA 溶液 (80 µM)。在25摄氏度的混合物中孵育30分钟, 保护光线。</li>
	<li>通过加入胰蛋白酶 (1:100 蛋白酶到基质蛋白比值 [w/w]) 来消化 GFP-E90DOPA 样品, 并在37摄氏度的4小时内孵化反应混合物。</li>
	<li>采用 C18自旋柱的标准脱盐法纯化消化肽样品。</li>
	<li>以 alpha-cyano-4-hydroxycinnamic 酸 (CHCA) 为基质23, 通过对基质辅助激光解吸/电离飞行时间质谱 (MALDI) 的报告协议, 对消化肽进行分析。</li>
</ol>

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I., 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.">Continuous directed evolution of aminoacyl-tRNA synthetases.</a> Nature Chemical Biology. 13 (12), 1253-1260 (2017).</li><li>Guo, J., Melancon, C. E., Lee, H. S., Groff, D., 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=Evolution+of+amber+suppressor+tRNAs+for+efficient+bacterial+production+of+proteins+containing+nonnatural+amino+acids.">Evolution of amber suppressor tRNAs for efficient bacterial production of proteins containing nonnatural amino acids.</a> Angewandte Chemie International Edition. 48 (48), 9148-9151 (2009).</li><li>Lee, 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=A+facile+strategy+for+selective+phosphoserine+incorporation+in+histones.">A facile strategy for selective phosphoserine incorporation in histones.</a> Angewandte Chemie International Edition. 52 (22), 5771-5775 (2013).</li><li>Neumann, H., Wang, K., Davis, L., Garcia-Alai, M., 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=Encoding+multiple+unnatural+amino+acids+via+evolution+of+a+quadruplet-decoding+ribosome.">Encoding multiple unnatural amino acids via evolution of a quadruplet-decoding ribosome.</a> Nature. 464 (7287), 441-444 (2010).</li><li>Lang, K., 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=Bioorthogonal+reactions+for+labeling+proteins.">Bioorthogonal reactions for labeling proteins.</a> ACS Chemical Biology. 9 (1), 16-20 (2014).</li><li>Lang, K., Chin, J. 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作者没有什么可透露的。

在本议定书中, 介绍了巴胺的生物合成和直接加入。这种方法中使用的细菌细胞可以合成额外的氨基酸, 并将其用作蛋白质合成的非自然构造块。非自然氨基酸的遗传融合一直是生物进化的关键技术, 具有扩展的遗传密码。然而, 这种方法在技术上是不完整的, 正在修改, 以提高整合效率, 并尽量减少扰动的内源翻译系统。最近, 该方法取得了重大进展, 通过重新分配密码子 24,</su...

巴胺是一种氨基酸, 涉及动物和植物中儿茶酚的生物合成。该氨基酸由酪氨酸羟化酶和分子氧 (O2)1合成 Tyr。由于多巴是一大胺的前驱体, 可以渗透血脑屏障, 它已用于治疗帕金森病2。巴比在贻贝黏附蛋白 (地图) 也被发现, 负责贻贝的胶粘剂物产在湿的情况3,4,5,6,7。Tyr 最初是在地图上发现了几巴的位置编码的, 然后被 tyrosinases8、9转换成了巴比。多巴由于其具有有趣的生化特性, 在各种应用中得到了应用。多巴二羟基组化学上易氧化, 氨基酸容易转化为 l-dopaquinone, 黑色素的前驱体。由于其高 electrophilicity, l-dopaquinone 及其衍生物已被用于交联和共轭与硫醇和胺10,11,12,13。12-醌还可以作为 cycloaddition 反应的二烯, 并已用于 bioconjugation 由应变促进氧化控制 cyclooctyne-1,2-quinone (SPOCQ) cycloaddition14。此外, 二羟基集团可以螯合金属离子, 如 Fe3 +和铜2 +, 和含有多巴的蛋白质已用于药物输送和金属离子传感15,16。
多巴通过使用正交氨酰-tRNA (aa-tRNA) 和氨酰-tRNA 合成酶 (aaRS) 对17 , 并用于蛋白质共轭和交联10,11, 被基因纳入蛋白质,12,13。在本报告中, 介绍了从廉价的起始材料中引入 biosynthesized 的实验结果和协议, 以及它在 bioconjugation 中的应用。biosynthesized 使用 TPL, 从邻苯二酚, 丙酮酸和氨的大肠杆菌开始。biosynthesized 多巴是通过表达一个进化的 aa-tRNA 和 aaRS 对巴比直接纳入蛋白质。此外, 含有多巴的 biosynthesized 蛋白与荧光探针结合, 并交联以产生蛋白质寡聚物。这项协议将是有用的蛋白质科学家, biosynthesize 突变蛋白含有一巴和共轭蛋白质与生化探针或药物的工业和制药应用。

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			<td height="21" style="height:21px;width:596px;">1. Plasmid Construction</td>
			<td style="width:167px;"></td>
			<td style="width:133px;"></td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:596px;">Plasmid pBAD-dual-TPL-GFP-E90TAG</td>
			<td style="width:167px;"></td>
			<td style="width:133px;"></td>
			<td>optionally contain the amber stop codon(TAG) at a desired position. Ko, W. et al. Efficient and Site-Specific Antibody Labeling by Strain-promoted Azide-Alkyne Cycloaddition. BKCS. 36 (9), 2352-2354, doi: 10.1002/bkcs.10423, (2015)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">Plasmid pEvol-DHPRS2</td>
			<td style="width:167px;"></td>
			<td style="width:133px;"></td>
			<td>1. Young, T. S., Ahmad, I., Yin, J. A., and Schultz, P. G. An enhanced system for unnatural amino acid mutagenesis in E. coli. J. Mol. Biol. 395 (2), 361-374, doi: 10.1016/j.jmb.2009.10.030, (2010) 2. Kim, S., Sung, B. H., Kim, S. C., Lee, H. S. Genetic incorporation of l-dihydroxyphenylalanine (DOPA) biosynthesized by a tyrosine phenol-lyase. Chem. Commun. 54 (24), 3002-3005, doi: 10.1039/c8cc00281a (2018).</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">DH10&#946;</td>
			<td style="width:167px;">Invitrogen</td>
			<td style="width:133px;">C6400-03</td>
			<td>Expression Host</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">Plasmid Mini-prep kit</td>
			<td style="width:167px;">Nucleogen</td>
			<td style="width:133px;">5112</td>
			<td>200/pack</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">Agarose</td>
			<td style="width:167px;">Intron biotechnology</td>
			<td style="width:133px;">32034</td>
			<td>500 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">Ethidium bromide</td>
			<td style="width:167px;">Alfa Aesar</td>
			<td style="width:133px;">L07482</td>
			<td>1 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">LB Broth</td>
			<td style="width:167px;">BD Difco</td>
			<td style="width:133px;">244620</td>
			<td>500 g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:596px;">2. Culture preparation</td>
			<td style="width:167px;"></td>
			<td style="width:133px;"></td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:596px;">2.1) Electroporation</td>
			<td style="width:167px;"></td>
			<td style="width:133px;"></td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">Micro pulser&#160;</td>
			<td style="width:167px;">BIO-RAD</td>
			<td style="width:133px;">165-2100</td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">Micro pulser cuvette</td>
			<td style="width:167px;">BIO-RAD</td>
			<td style="width:133px;">165-2089</td>
			<td style="width:339px;">0.1 cm electrode gap, pkg. of 50</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">Ampicillin Sodium</td>
			<td style="width:167px;">Wako</td>
			<td style="width:133px;">018-10372</td>
			<td>25 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">Chloramphenicol</td>
			<td style="width:167px;">Alfa Aesar</td>
			<td style="width:133px;">B20841</td>
			<td>25 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">Agar</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">214230</td>
			<td>500 g</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:596px;">SOC medium</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:133px;">S1797</td>
			<td>100 mL</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:596px;">3. Expression and Purification of GFP-E90DOPA by biosynthetic system</td>
			<td style="width:167px;"></td>
			<td style="width:133px;"></td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:596px;">3.1 Expression of GFP-E90DOPA by biosynthetic system</td>
			<td style="width:167px;"></td>
			<td style="width:133px;"></td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">L(+)-Arabinose, 99%</td>
			<td style="width:167px;">Acros</td>
			<td style="width:133px;">104981000</td>
			<td>100 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">Pyrocatechol, 99%</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">P1387</td>
			<td>25 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">Ammonium sulfate, 99%</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">A0943</td>
			<td>500 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">pyruvic acid, 98%</td>
			<td style="width:167px;">Alfa Aesar</td>
			<td style="width:133px;">A13875</td>
			<td>100 g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:596px;">Sodium phosphate dibasic, anhydrous, 99%</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">S0891</td>
			<td>1 kg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">Potassium phophate, monobasic, 99%</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">P1127</td>
			<td>1 kg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">Magnesium sulfate, anhydrous, 99%</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">M0146</td>
			<td>1 kg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">D(+)-Glucose, anhydrous, 99%</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">D0092</td>
			<td>500 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">Glycerol, 99%</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">G0269</td>
			<td>1 kg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">Trace metal mix a5 with co</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:133px;">92949</td>
			<td>25 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">L-Proline, 99%</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">P1257</td>
			<td>25 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">L-Phenylalanine, 98.5%</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">P1982</td>
			<td>25 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">L-Tryptophane</td>
			<td style="width:167px;">JUNSEI</td>
			<td style="width:133px;">49550-0310</td>
			<td>25 g</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:596px;">L-Arginine, 98%</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">A1149</td>
			<td>25 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">L-Glutamine, 98%</td>
			<td style="width:167px;">JUNSEI</td>
			<td style="width:133px;">27340-0310</td>
			<td>25 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">L-Asparagine monohydrate, 99%</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">A1198</td>
			<td>25 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">L-Methionine</td>
			<td style="width:167px;">JUNSEI</td>
			<td style="width:133px;">73190-0410</td>
			<td>25 g</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:596px;">L-Histidine hydrochloride monohydrate, 99%</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">H0604</td>
			<td>25 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">L-Threonine, 99%</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">T2938</td>
			<td>25 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">L-Leucine</td>
			<td style="width:167px;">JUNSEI</td>
			<td style="width:133px;">87070-0310</td>
			<td>25 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">Glycine, 99%</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">G0286</td>
			<td>25 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">L-Glutamic acid, 99%</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">G0233</td>
			<td>25 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">L-Alanine, 99%</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">A1543</td>
			<td>25 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">L-Isoleucine, 99%</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">I1049</td>
			<td>25 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">L-Valine, 99%</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">V0088</td>
			<td>25 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">L-Serine</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">S2447</td>
			<td>25 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">L-Aspartic acid</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">A1205</td>
			<td>25 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">L-Lysine monohydrochloride, 99%</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">L0592</td>
			<td>25 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">3.2 Cell lysis</td>
			<td style="width:167px;"></td>
			<td style="width:133px;"></td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">Imidazole, 99%</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">I0578</td>
			<td>1kg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">Sodium phosphate monobasic, 98%</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">S0919</td>
			<td>1 kg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">Sodium Chloride, 99%</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">S2907</td>
			<td>1 kg</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:596px;">Ultrasonic Processor - 150 microliters to 150 milliliters</td>
			<td style="width:167px;">SONIC &#38; MATERIALS</td>
			<td style="width:133px;">VCX130</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">3.3 Ni-NTA Affinity Chromatography</td>
			<td style="width:167px;"></td>
			<td style="width:133px;"></td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">Ni-NTA resin</td>
			<td style="width:167px;">QIAGEN</td>
			<td style="width:133px;">30210</td>
			<td>25 mL</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:596px;">Polypropylene column</td>
			<td style="width:167px;">QIAGEN</td>
			<td style="width:133px;">34924</td>
			<td>50/pack, 1 mL capacity</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:596px;">4. Oligomerization of Purified GFP-E90DOPA&#160;</td>
			<td style="width:167px;"></td>
			<td style="width:133px;"></td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:596px;">Sodium periodate, 99.8&#38;</td>
			<td style="width:167px;">Acros</td>
			<td style="width:133px;">419610050</td>
			<td>5 g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:596px;">5. Conjugation of GFP-E90DOPA with an Alkyne Probe by Strain-Promoted Oxidation-Controlled Cyclooctyne&#8211;1,2-Quinone Cycloaddition (SPOCQ)&#160;</td>
			<td style="width:167px;"></td>
			<td style="width:133px;"></td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:596px;">Cy5.5-ADIBO&#160;</td>
			<td style="width:167px;">FutureChem</td>
			<td style="width:133px;">FC-6119</td>
			<td>1mg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">6. Purification of Labeled GFP</td>
			<td style="width:167px;"></td>
			<td style="width:133px;"></td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:596px;">Amicon Ultra 0.5 mL Centrifugal Filters</td>
			<td style="width:167px;">MILLIPORE</td>
			<td style="width:133px;">UFC500396</td>
			<td>96/pack, 500ul capacity</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:596px;">7. SDS-PAGE Analysis and Fluorescence Gel Scanning</td>
			<td style="width:167px;"></td>
			<td style="width:133px;"></td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">1,4-Dithio-DL-threitol, DTT, 99.5 %</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:133px;">10708984001</td>
			<td>10 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">NuPAGE LDS Sample Buffer, 4X</td>
			<td style="width:167px;">Thermofisher</td>
			<td style="width:133px;">NP0007</td>
			<td>10 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">MES running buffer</td>
			<td style="width:167px;">Thermofisher</td>
			<td style="width:133px;">NP0002</td>
			<td>500 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">Nupage Novex 4-12% SDS PAGE gels</td>
			<td style="width:167px;">Thermofisher</td>
			<td style="width:133px;">NO0321</td>
			<td>12 well</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">Coomassie Brilliant Blue R-250</td>
			<td style="width:167px;">Wako</td>
			<td style="width:133px;">031-17922</td>
			<td>25 g</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:596px;">G:BOX Chemi Fluorescent &#38; Chemiluminescent Imaging System</td>
			<td style="width:167px;">Syngene</td>
			<td style="width:133px;"></td>
			<td>G BOX Chemi XT4</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:596px;">8. MALDI-TOF MS analysis by Trypsin Digestion</td>
			<td style="width:167px;"></td>
			<td style="width:133px;"></td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:596px;">8.1 Preparation of the digested peptide sample by trypsin digestion</td>
			<td style="width:167px;"></td>
			<td style="width:133px;"></td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">Tris(hydroxymethyl)aminomethane, 99%</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">T1351</td>
			<td>500 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">Hydrochloric acid, 35~37%</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">H0256</td>
			<td>500 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">Dodecyl sulfate sodium salt, 85%</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">D1070</td>
			<td>250 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">Iodoacetamide</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:133px;">I6125</td>
			<td>5 g</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:596px;">Trypsin Protease, MS Grade</td>
			<td style="width:167px;">Thermofisher</td>
			<td style="width:133px;">90057</td>
			<td>5 x 20 &#181;g/pack</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">C-18 spin columns</td>
			<td style="width:167px;">Thermofisher</td>
			<td style="width:133px;">89870</td>
			<td>25/pack, 200 &#181;L capacity</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:596px;">8.2 Analysis of the digested peptide by MALDI-TOF</td>
			<td style="width:167px;"></td>
			<td style="width:133px;"></td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">Acetonitirile, 99.5%</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">A0125</td>
			<td>500 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">&#945;-Cyano-4-hydroxycinnamic acid</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:133px;">C2020</td>
			<td>10 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">Trifluoroacetic acid, 99%</td>
			<td style="width:167px;">SAMCHUN</td>
			<td style="width:133px;">T1666</td>
			<td>100 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">MTP 384 target plate ground steel BC targets</td>
			<td style="width:167px;">Bruker</td>
			<td style="width:133px;">8280784</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:596px;">Bruker Autoflex Speed MALDI-TOF mass spectrometer</td>
			<td style="width:167px;">Bruker</td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

genetic incorporation of biosynthesized l-dihydroxyphenylalanine (dopa) and its application to protein conjugation

l-dihydroxyphenylalanine (多巴) 是在动物和植物中儿茶酚胺的生物合成中发现的一种氨基酸。由于氨基酸具有特殊的生化特性, 在生物化学应用中具有多种用途。本报告描述了 biosynthesized 巴的遗传合并协议及其在蛋白质共轭中的应用。多巴是由邻苯二酚、丙酮酸和氨的酪氨酸酚裂解酶 (TPL) biosynthesized 的, 而氨基酸则采用进化后的氨酰-tRNA 和氨酰-tRNA 合成产物对的遗传法直接纳入蛋白质中。这种直接的并网系统有效地吸收了多巴与其他天然氨基酸的很少结合, 并具有更好的蛋白质产量比以前的多巴基因合并系统。多巴多巴蛋白结合具有高效性和特异性, 并显示其对各种应用的实用性。该议定书为蛋白质科学家提供了在所需地点对含有巴胺的突变蛋白进行有效生物合成的详细程序, 以及它们用于工业和制药应用的共轭。

图 1显示了从 TPL 直接并入 biosynthesized 的表达系统。进化的 aa-tRNA 和 aaRS 对的基因被放置在质粒中, 而 gfp 基因 (GFP-E90TAG) 包含琥珀密码子在位置90位于另一个质粒中, 以评估多巴的加入 gfp 荧光。TPL 基因被放置在含有 GFP 基因和组成性表达的同一个表达质粒中, 以最大程度地提高巴比生物合成的产量。利用该表达系统筛选了巴比生物合成的最佳条件。本...

Watch this Scientific Journal Video about Genetic Incorporation of Biosynthesized L-dihydroxyphenylalanine DOPA and Its Application to Protein Conjugation at JoVE.com

Genetic Incorporation of Biosynthesized L-dihydroxyphenylalanine DOPA and Its Application to Protein Conjugation

Biosynthesized l-dihydroxyphenylalanine (多巴) 的遗传合并及其在蛋白质共轭中的应用

在此, 我们提出了一种从简单的起始材料中 dihydroxyphenylalanine biosynthesized 的遗传方法及其在蛋白质共轭中的应用的协议。

L-dihydroxyphenylalanine (DOPA) is an amino acid found in the biosynthesis of catecholamines in animals and plants. Because of its particular biochemical ...

genetic-incorporation-biosynthesized-l-dihydroxyphenylalanine-dopa

Research

JoVE Journal

Biochemistry

Genetic Incorporation of Biosynthesized L-dihydroxyphenylalanine (DOPA) and Its Application to Protein Conjugation

7.8K 次观看.  Sogang University. 在此, 我们提出了一种从简单的起始材料中 dihydroxyphenylalanine biosynthesized 的遗传方法及其在蛋白质共轭中的应用的协议。

视频: Genetic Incorporation of Biosynthesized L-dihydroxyphenylalanine DOPA and Its Application to Protein Conjugation

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating these studies, reincorporation of detergent-solubilized membrane proteins into liposomes is a stochastic process where protein topology is impossible to enforce. This paper offers an alternative method to these challenging techniques that utilizes a liposome-based scaffold. Protein solubility is enhanced by deletion of the transmembrane domain, and these amino acids are replaced with a tethering moiety, such as a His-tag. This tether interacts with an anchoring group (Ni2+ coordinated by nitrilotriacetic acid (NTA(Ni2+)) for His-tagged proteins), which enforces a uniform protein topology at the surface of the liposome. An example is presented wherein the interaction between Dynamin-related protein 1 (Drp1) with an integral membrane protein, Mitochondrial Fission Factor (Mff), was investigated using this scaffold liposome method. In this work, we have demonstrated the ability of Mff to efficiently recruit soluble Drp1 to the surface of liposomes, which stimulated its GTPase activity. Moreover, Drp1 was able to tubulate the Mff-decorated lipid template in the presence of specific lipids. This example demonstrates the effectiveness of scaffold liposomes using structural and functional assays and highlights the role of Mff in regulating Drp1 activity.

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment.

使用脚手架脂质体重构脂质近端蛋白质 - 蛋白质相互作用<em&gt;体外</em

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

科研

生物化学

A Protein Preparation Method for the High-throughput Identification of Proteins Interacting with a Nuclear Cofactor Using LC-MS/MS Analysis

Transcriptional coregulators are vital to the efficient transcriptional regulation of nuclear chromatin structure. Coregulators play a variety of roles in regulating transcription. These include the direct interaction with transcription factors, the covalent modification of histones and other proteins, and the occasional chromatin conformation alteration. Accordingly, establishing relatively quick methods for identifying proteins that interact within this network is crucial to enhancing our understanding of the underlying regulatory mechanisms. LC-MS/MS-mediated protein binding partner identification is a validated technique used to analyze protein-protein interactions. By immunoprecipitating a previously-identified member of a protein complex with an antibody (occasionally with an antibody for a tagged protein), it is possible to identify its unknown protein interactions via mass spectrometry analysis. Here, we present a method of protein preparation for the LC-MS/MS-mediated high-throughput identification of protein interactions involving nuclear cofactors and their binding partners. This method allows for a better understanding of the transcriptional regulatory mechanisms of the targeted nuclear factors.

We have established a method for the purification of coregulatory interaction proteins using the LC-MS/MS system.

A蛋白制备方法在蛋白质与核余子交互使用LC-MS / MS分析的高通量鉴定

Transcriptional coregulators are vital to the efficient transcriptional regulation of nuclear chromatin structure. Coregulators play a variety of roles in ...

Preparation of Keratin Hydrolysate from Chicken Feathers and Its Application in Cosmetics

Keratin hydrolysates (KHs) are established standard components in hair cosmetics. Understanding the moisturizing effects of KH is advantageous for skin-care cosmetics. The goals of the protocol are: (1) to process chicken feathers into KH by alkaline-enzymatic hydrolysis and purify it by dialysis, and (2) to test if adding KH into an ointment base (OB) increases hydration of the skin and improves skin barrier function by diminishing transepidermal water loss (TEWL). During alkaline-enzymatic hydrolysis feathers are first incubated at a higher temperature in an alkaline environment and then, under mild conditions, hydrolyzed with proteolytic enzyme. The solution of KH is dialyzed, vacuum dried, and milled to a fine powder. Cosmetic formulations comprising from oil in water emulsion (O/W) containing 2, 4, and 6 weight% of KH (based on the weight of the OB) are prepared. Testing the moisturizing properties of KH is carried out on 10 men and 10 women at time intervals of 1, 2, 3, 4, 24, and 48 h. Tested formulations are spread at degreased volar forearm sites. The skin hydration of stratum corneum (SC) is assessed by measuring capacitance of the skin, which is one of the most world-wide used and simple methods. TEWL is based on measuring the quantity of water transported per a defined area and period of time from the skin. Both methods are fully non-invasive. KH makes for an excellent occlusive; depending on the addition of KH into OB, it brings about a 30% reduction in TEWL after application. KH also functions as a humectant, as it binds water from the lower layers of the epidermis to the SC; at the optimum KH addition in the OB, up to 19% rise in hydration in men and 22% rise in women occurs.

The goal of the protocol is to prepare keratin hydrolysate from chicken feathers by alkaline-enzymatic hydrolysis and to test whether adding keratin hydrolysate into a cosmetics ointment base improves skin barrier function (heightening hydration and diminishing transepidermal water loss). Tests are conducted on men and woman volunteers.

鸡羽毛角蛋白水解物的制备及其在化妆品中的应用

Keratin hydrolysates (KHs) are established standard components in hair cosmetics. Understanding the moisturizing effects of KH is advantageous for ...

Biotinylated Cell-penetrating Peptides to Study Intracellular Protein-protein Interactions

Here we present a protocol to study intracellular protein-protein interactions that is based on the widely used biotin-avidin pull-down system. The modification presented includes the combination of this technique with cell-penetrating sequences. We propose to design cell-penetrating baits that can be incubated with living cells instead of cell lysates and therefore the interactions found will reflect those that occur within the intracellular context. Connexin43 (Cx43), a protein that forms gap junction channels and hemichannels is down-regulated in high-grade gliomas. The Cx43 region comprising amino acids 266-283 is responsible for the inhibition of the oncogenic activity of c-Src in glioma cells. Here we use TAT as the cell-penetrating sequence, biotin as the pull-down tag and the region of Cx43 comprised between amino acids 266-283 as the target to find intracellular interactions in the hard-to-transfect human glioma stem cells. One of the limitations of the proposed method is that the molecule used as bait could fail to fold properly and, consequently, the interactions found could not be associated with the effect. However, this method can be especially interesting for the interactions involved in signal transduction pathways because they are usually carried out by intrinsically disordered regions and, therefore, they do not require an ordered folding. In addition, one of the advantages of the proposed method is that the relevance of each residue on the interaction can be easily studied. This is a modular system; therefore, other cell-penetrating sequences, other tags, and other intracellular targets can be employed. Finally, the scope of this protocol is far beyond protein-protein interaction because this system can be applied to other bioactive cargoes such as RNA sequences, nanoparticles, viruses or any molecule that can be transduced with cell-penetrating sequences and fused to pull-down tags to study their intracellular mechanism of action.

This is a protocol to study intracellular protein-protein interactions based on the biotin-avidin pull-down system with the novelty of combining cell-penetrating sequences. The main advantage is that the target sequence is incubated with living cells instead of cell lysates and therefore the interactions will occur within the cellular context.

化细胞穿透肽研究胞内蛋白-蛋白质相互作用

Here we present a protocol to study intracellular protein-protein interactions that is based on the widely used biotin-avidin pull-down system. The ...

High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing approaches suffer from significant limitations, we have developed a new method for determining TF-DNA binding affinities with high sensitivity on a large scale. The assay relies on the established fluorescence anisotropy (FA) principle but introduces important technical improvements. First, we measure a full FA competitive titration curve in a single well by incorporating TF and a fluorescently labeled reference DNA in a porous agarose gel matrix. Unlabeled DNA oligomer is loaded on the top as a competitor and, through diffusion, forms a spatio-temporal gradient. The resulting FA gradient is then read out using a customized epifluorescence microscope setup. This improved setup greatly increases the sensitivity of FA signal detection, allowing both weak and strong binding to be reliably quantified, even for molecules of similar molecular weights. In this fashion, we can measure one titration curve per well of a multi-well plate, and through a fitting procedure, we can extract both the absolute dissociation constant (KD) and active protein concentration. By testing all single-point mutation variants of a given consensus binding sequence, we can survey the entire binding specificity landscape of a TF, typically on a single plate. The resulting position weight matrices (PWMs) outperform those derived from other methods in predicting in vivo TF occupancy. Here, we present a detailed guide for implementing HiP-FA on a conventional automated fluorescent microscope and the data analysis pipeline.

Here we present a novel method for determining binding affinities at equilibrium and in solution with high sensitivity on a large scale. This improves the quantitative analysis of transcription factor-DNA binding. The method is based on automated fluorescence anisotropy measurements in a controlled delivery system.

荧光显微镜竞争滴定法测定转录机-dna 结合感的高灵敏度

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing ...

Analysis of Protein Folding, Transport, and Degradation in Living Cells by Radioactive Pulse Chase

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and the degradation of target proteins in live cells. By using short (pulse) radiolabeling times (&lt;30 min) and tightly controlled chase times, it is possible to label only a small fraction of the total protein pool and follow its folding. When combined with nonreducing/reducing SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoprecipitation with (conformation-specific) antibodies, folding processes can be examined in great detail. This system has been used to analyze the folding of proteins with a huge variation in properties such as soluble proteins, single and multi-pass transmembrane proteins, heavily N- and O-glycosylated proteins, and proteins with and without extensive disulfide bonding. Pulse-chase methods are the basis of kinetic studies into a range of additional features, including co- and posttranslational modifications, oligomerization, and polymerization, essentially allowing the analysis of a protein from birth to death. Pulse-chase studies on protein folding are complementary with other biochemical and biophysical methods for studying proteins in vitro by providing increased temporal resolution and physiological information. The methods as described within this paper are adapted easily to study the folding of almost any protein that can be expressed in mammalian or insect-cell systems.

Here we describe a protocol for a general pulse-chase method that allows the kinetic analysis of folding, transport, and degradation of proteins to be followed in live cells.

放射性脉冲追逐对活细胞蛋白质折叠、迁移和降解的分析

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and ...

Application of Biolayer Interferometry (BLI) for Studying Protein-Protein Interactions in Transcription

A transcription factor&#160;(TF) is a protein that regulates gene expression by interacting with the RNA polymerase, another TF, and/or template DNA. GrgA is a novel transcription activator found specifically in the obligate intracellular bacterial pathogen Chlamydia. Protein pulldown assays using affinity beads have revealed that GrgA binds two &#963; factors, namely &#963;66 and &#963;28, which recognize different sets of promoters for genes whose products are differentially required at developmental stages. We have used BLI to confirm and further characterize the interactions. BLI demonstrates several advantages over pulldown: 1) It reveals real-time association and dissociation between binding partners, 2) It generates quantitative kinetic parameters, and 3) It can detect bindings that pulldown assays often fail to detect. These characteristics have enabled us to deduce the physiological roles of GrgA in gene expression regulation in Chlamydia, and possible detailed interaction mechanisms. We envision that this relatively affordable technology can be extremely useful for studying transcription and other biological processes.

Application of Biolayer Interferometry BLI for Studying Protein-Protein Interactions in Transcription

Interactions of transcription factors (TFs) with the RNA polymerase are usually studied using pulldown assays. We apply a Biolayer Interferometry (BLI) technology to characterize the interaction of GrgA with the chlamydial RNA polymerase. Compared to pulldown assays, BLI detects real-time association and dissociation, offers higher sensitivity, and is highly quantitative.

生物层干涉测量(BLI)在转录中研究蛋白质-蛋白质相互作用的应用

A transcription factor&#160;(TF) is a protein that regulates gene expression by interacting with the RNA polymerase, another TF, and/or template DNA. GrgA ...

PeptiQuick, a One-Step Incorporation of Membrane Proteins into Biotinylated Peptidiscs for Streamlined Protein Binding Assays

Membrane proteins, including transporters, channels, and receptors, constitute nearly one-fourth of the cellular proteome and over half of current drug targets. Yet, a major barrier to their characterization and exploitation in academic or industrial settings is that most biochemical, biophysical, and drug screening strategies require these proteins to be in a water-soluble state. Our laboratory recently developed the peptidisc, a membrane mimetic offering a &#8220;one-size-fits-all&#8221; approach to the problem of membrane protein solubility. We present here a streamlined protocol that combines protein purification and peptidisc reconstitution in a single chromatographic step. This workflow, termed PeptiQuick, allows for bypassing dialysis and incubation with polystyrene beads, thereby greatly reducing exposure to detergent, protein denaturation, and sample loss. When PeptiQuick is performed with biotinylated scaffolds, the preparation can be directly attached to streptavidin-coated surfaces. There is no need to biotinylate or modify the membrane protein target. PeptiQuick is showcased here with the membrane receptor FhuA and antimicrobial ligand colicin M, using biolayer interferometry to determine the precise kinetics of their interaction. It is concluded that PeptiQuick is a convenient way to prepare and analyze membrane protein-ligand interactions within one&#160;day in a detergent-free environment.

We present a method that combines membrane protein purification and reconstitution into peptidiscs in a single chromatographic step. Biotinylated scaffolds are used for direct surface attachment and measurement of protein-ligand interactions via biolayer interferometry.

PeptiQuick，将膜蛋白一步进生物仿入肽，用于简化蛋白质结合测定

Membrane proteins, including transporters, channels, and receptors, constitute nearly one-fourth of the cellular proteome and over half of current drug ...

Creating Highly Specific Chemically Induced Protein Dimerization Systems by Stepwise Phage Selection of a Combinatorial Single-Domain Antibody Library

Protein dimerization events that occur only in the presence of a small-molecule ligand enable the development of small-molecule biosensors for the dissection and manipulation of biological pathways. Currently, only a limited number of chemically induced dimerization (CID) systems exist and engineering new ones with desired sensitivity and selectivity for specific small-molecule ligands remains a challenge in the field of protein engineering. We here describe a high throughput screening method, combinatorial binders-enabled selection of CID (COMBINES-CID), for the de novo engineering of CID systems applicable to a large variety of ligands. This method uses the two-step selection of a phage-displayed combinatorial nanobody library to obtain 1) &#34;anchor binders&#34; that first bind to a ligand of interest and then 2) &#34;dimerization binders&#34; that only bind to anchor binder-ligand complexes. To select anchor binders, a combinatorial library of over 109 complementarity-determining region (CDR)-randomized nanobodies is screened with a biotinylated ligand and hits are validated with the unlabeled ligand by bio-layer interferometry (BLI). To obtain dimerization binders, the nanobody library is screened with anchor binder-ligand complexes as targets for positive screening and the unbound anchor binders for negative screening. COMBINES-CID is broadly applicable to select CID binders with other immunoglobulin, non-immunoglobulin, or computationally designed scaffolds to create biosensors for in vitro and in vivo detection of drugs, metabolites, signaling molecules, etc.

Creating chemically induced protein dimerization systems with desired affinity and specificity for any given small molecule ligand would have many biological sensing and actuation applications. Here, we describe an efficient, generalizable method for de novo engineering of chemically induced dimerization systems via the stepwise selection of a phage-displayed combinatorial single-domain antibody library.

通过组合单域抗体库的分步噬菌体选择创建高度特异性化学诱导蛋白分解系统

Protein dimerization events that occur only in the presence of a small-molecule ligand enable the development of small-molecule biosensors for the ...

JUMPn: A Streamlined Application for Protein Co-Expression Clustering and Network Analysis in Proteomics

With recent advances in mass spectrometry-based proteomics technologies, deep profiling of hundreds of proteomes has become increasingly feasible. However, deriving biological insights from such valuable datasets is challenging. Here we introduce a systems biology-based software JUMPn, and its associated protocol to organize the proteome into protein co-expression clusters across samples and protein-protein interaction (PPI) networks connected by modules (e.g., protein complexes). Using the R/Shiny platform, the JUMPn software streamlines the analysis of co-expression clustering, pathway enrichment, and PPI module detection, with integrated data visualization and a user-friendly interface. The main steps of the protocol include installation of the JUMPn software, the definition of differentially expressed proteins or the (dys)regulated proteome, determination of meaningful co-expression clusters and PPI modules, and result visualization. While the protocol is demonstrated using an isobaric labeling-based proteome profile, JUMPn is generally applicable to a wide range of quantitative datasets (e.g., label-free proteomics). The JUMPn software and protocol thus provide a powerful tool to facilitate biological interpretation in quantitative proteomics.

We present a systems biology tool JUMPn to perform and visualize network analysis for quantitative proteomics data, with a detailed protocol including data pre-processing, co-expression clustering, pathway enrichment, and protein-protein interaction network analysis.

Biosynthesized l-dihydroxyphenylalanine (多巴) 的遗传合并及其在蛋白质共轭中的应用

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