Name: biosynthesis of a flavonol from a flavanone by establishing a one-pot bienzymatic cascade
Uploaded: 2019-08-14T12:30:00
Description: Watch this Scientific Journal Video about Biosynthesis of a Flavonol from a Flavanone by Establishing a One-pot Bienzymatic Cascade at JoVE.com

这项工作由扬州大学特聘教授创业基金、江苏省特聘教授创业基金、江苏省六大人才高峰项目(授权2014-SWYY-016)和江苏省资助项目资助。江苏省高等学校(兽医)重点学术项目发展。感谢扬州大学黄酮类化合物的HPLC和MS分析测试中心。

1. 从植物组织中分离总RNA26,27
<ol><li>使植物组织同质化。<ol>
<li>收集100毫克的新鲜植物组织(例如,4周大的幼苗从阿拉伯拟南芥)。用液氮冷冻组织和虫子和砂浆,然后将组织研磨成粉末。</li>
		<li>将1mL的RNA分离试剂(见材料表)加入砂浆中。试剂将立即冷冻。当冷冻试剂熔化时,将组织样品与虫子同质化。</li>
		<li>将均质转移到1.5-mL管中,?...

<ol>
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Species Growing in Turkey.</a> Molecules. 20 (10), 17976-18000 (2015).</li><li>Yang, R. Y., Lin, S., Kuo, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Content+and+distribution+of+flavonoids+among+91+edible+plant+species.">Content and distribution of flavonoids among 91 edible plant species.</a> Asia Pacific Journal of Clinical Nutrition. 17, 275-279 (2008).</li><li>Tang, L. J., Zhang, S. F., Yang, J. Z., Gao, W. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+Synthetic+Methods+of+Flavones.">New Synthetic Methods of Flavones.</a> Chinese Journal of Organic Chemistry. 24 (8), 882-889 (2004).</li><li>Lu, Y. H., 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=Synthesis+of+luteolin+and+kaempferol+(author's+transl).">Synthesis of luteolin and kaempferol (author's transl).</a> Yao Xue Xue Bao. 15 (8), 477-481 (1980).</li><li>Zhang, Z., 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=Development+and+Optimization+of+an+In+vitro+Multienzyme+Synthetic+System+for+Production+of+Kaempferol+from+Naringenin.">Development and Optimization of an In vitro Multienzyme Synthetic System for Production of Kaempferol from Naringenin.</a> Journal of Agricultural and Food Chemistry. 66 (31), 8272-8279 (2018).</li><li>Malla, S., Pandey, R. P., Kim, B. G., Sohng, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regiospecific+modifications+of+naringenin+for+astragalin+production+in+Escherichia+coli.">Regiospecific modifications of naringenin for astragalin production in Escherichia coli.</a> Biotechnology and Bioengineering. 110 (9), 2525-2535 (2013).</li><li>Zhu, S., Wu, J., Du, G., Zhou, J., Chen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+synthesis+of+eriodictyol+from+L-tyrosine+in+Escherichia+coli.">Efficient synthesis of eriodictyol from L-tyrosine in Escherichia coli.</a> Applied and Environmental Microbiology. 80 (10), 3072-3080 (2014).</li><li>Trantas, E., Panopoulos, N., Ververidis, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+engineering+of+the+complete+pathway+leading+to+heterologous+biosynthesis+of+various+flavonoids+and+stilbenoids+in+Saccharomyces+cerevisiae.">Metabolic engineering of the complete pathway leading to heterologous biosynthesis of various flavonoids and stilbenoids in Saccharomyces cerevisiae.</a> Metabolic Engineering. 11 (6), 355-366 (2009).</li><li>Miyahisa, 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=Combinatorial+biosynthesis+of+flavones+and+flavonols+in+Escherichia+coli.">Combinatorial biosynthesis of flavones and flavonols in Escherichia coli.</a> Applied Microbiology and Biotechnology. 71 (1), 53-58 (2006).</li><li>Leonard, E., Yan, Y., Koffas, M. 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A colorful model for genetics, biochemistry, cell biology, and biotechnology.</a> Plant Physiology. 126 (2), 485-493 (2001).</li><li>Duan, L., 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=Biosynthesis+and+engineering+of+kaempferol+in+Saccharomyces+cerevisiae.">Biosynthesis and engineering of kaempferol in Saccharomyces cerevisiae.</a> Microbial Cell Factories. 16 (1), 165 (2017).</li><li>Cheng, Q., Xiang, L., Izumikawa, M., Meluzzi, D., Moore, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymatic+total+synthesis+of+enterocin+polyketides.">Enzymatic total synthesis of enterocin polyketides.</a> Nature Chemical Biology. 3 (9), 557-558 (2007).</li><li>Connolly, M. A., Clausen, P. A., Lazar, J. 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相当多的研究侧重于黄酮醇的衍生,因为它们在保健和食品工业中具有潜在的应用。然而,传统的植物提取使用有机溶剂和化学合成具有内在的缺点,限制了其在黄酮醇的生产中使用。在这里,我们报告一种通过建立体外双酶级联从一个锅中从黄酮中产生黄酮的详细方法。该协议的关键步骤是:1)获得具有高活性的纯重组酶,2)建立一锅双酶反应级联。一般来说,植物源基因在细菌中的表达倾向于形成内含?...

黄酮类是植物类黄酮的主要亚类,涉及植物发育和色素沉着1,2,3。更重要的是,这些化合物具有广泛的有益健康活性,如抗癌4、5、抗氧化6、抗炎7、抗肥胖8、抗高血压9,和记忆回忆属性10,导致大量研究这些植物衍生的二次代谢物。传统上,这些化合物主要来自使用有机溶剂的植物萃取物。然而,由于在植物11、12、13中含量极低,大多数黄酮醇的生产成本仍然很高,这限制了其在医疗保健和食品中的应用。行业。
在过去的几十年里,科学家们已经开发出许多方法来衍生黄酮类化合物14,15。然而,这些复杂分子的化学合成具有各种内在缺点16。它不仅需要毒试剂和极端反应条件,还需要许多步骤来产生靶向黄酮类化合物14、17。此外,这一策略的另一个重要挑战是活性类黄酮分子的手性合成。因此,通过化学合成16、17,在商业规模上生产类黄酮不是理想的策略。
最近,科学家已经开发出一种有前途的替代策略,通过工程微生物生产这些复杂的天然化合物,为黄酮类生物合成途径18,19,20,21,22,这已被成功破译在植物23。例如,Duan等人将一种生物合成途径引入萌芽的酵母糖精,以产生kaempferol(KMF)24。马拉等人通过引入黄烷3-羟基酶(f3h)、黄酮合成酶(fls1)和UDP葡萄糖:黄酮3-O-葡萄糖转移酶UGTT78K1基因,生产出一种甘基化黄酮黄酮。大肠杆菌BL21(DE3)17 .尽管有相当多的模式,但并非所有基因工程微生物都生产感兴趣的产品,因为细胞平台的复杂性,人工合成的基因元素和宿主之间的不兼容,抑制目标产品对宿主细胞的影响,以及工程细胞系统本身的不稳定性16。
黄酮类化合物生产的另一个有前途的替代策略是在体外建立多酶级联。程等人曾报告说,肠多基肽可以通过在一锅25中组装一个完整的酶通路而成功合成。这种无细胞合成策略规避了微生物生产工厂的限制,因此可以大量生产一些类黄酮。
最近,我们成功地开发了一种双酶合成系统,将纳林金(NRN)在一罐16中转化为KMF。在这里,我们将详细介绍此系统以及分析产品所涉及的方法。我们还提供了两个示例,使用该系统从 NRN 和曲霉素 (QRC) 从二恶极醇 (ERD) 生成 KMF。此外,我们还讨论了该方法的关键步骤以及黄酮类化合物生物合成的未来研究方向。

<table>
	<tbody>
		<tr>
			<td>2&#215; Pfu MasterMix</td>
			<td>Beijing CoWin Biotech Co., Ltd</td>
			<td>CW0717A</td>
			<td>PCR amplification of genes with high fidelity</td>
		</tr>
		<tr>
			<td>Agilent 1200 Series RRLC system with an Agilent 6460 Triple Quadrupole LC/MS system</td>
			<td>Agilent Technologies, Inc</td>
			<td>N/A</td>
			<td>an equipment for analysis of flavonoids by HPLC/MS</td>
		</tr>
		<tr>
			<td>Agilent MassHunter Workstation (version B.03.01)</td>
			<td>Agilent Technologies, Inc</td>
			<td>N/A</td>
			<td>a software for collection of the data from the Agilent 1200 Series RRLC system with an Agilent 6460 Triple Quadrupole LC/MS system</td>
		</tr>
		<tr>
			<td>dihydrokaempferol</td>
			<td>Sigma-Aldrich Co. LLC</td>
			<td>91216</td>
			<td>intermediate product for producing kaempferol from naringenin</td>
		</tr>
		<tr>
			<td>dihydroquercetin</td>
			<td>Sichuan Provincial Standard Substance Center for Chinese Herbal Medicine</td>
			<td>PCS0371</td>
			<td>intermediate product for producing quercetin from eriodictyol</td>
		</tr>
		<tr>
			<td>DNA Clean-up Kit</td>
			<td>Beijing CoWin Biotech Co., Ltd</td>
			<td>CW2301</td>
			<td>purification of PCR-amplified or gel-purified DNA</td>
		</tr>
		<tr>
			<td>eriodictyol</td>
			<td>Shanghai Yuan Ye Biotechnology Co., Ltd.</td>
			<td>B21160</td>
			<td>substrate for producing quercetin</td>
		</tr>
		<tr>
			<td>Escherichia coli BL21(DE3)</td>
			<td>Beijing CoWin Biotech Co., Ltd</td>
			<td>CW0809</td>
			<td>bacteria strain for expressing target genes</td>
		</tr>
		<tr>
			<td>Escherichia coli DH5&#945;</td>
			<td>Beijing CoWin Biotech Co., Ltd</td>
			<td>CW0808</td>
			<td>bacteria strain for plasmid proliferation</td>
		</tr>
		<tr>
			<td>FreeZone 1 Liter Benchtop Freeze-Dry System</td>
			<td>Labconco Corporation</td>
			<td>7740020</td>
			<td>an equipment for freeze-drying of flavonoids dissolved in organic solvent</td>
		</tr>
		<tr>
			<td>Gel Extraction Kit</td>
			<td>Beijing CoWin Biotech Co., Ltd</td>
			<td>CW2302</td>
			<td>purification of a DNA band from an agarose gel</td>
		</tr>
		<tr>
			<td>Gel Imaging System</td>
			<td>Shanghai Tanon Science &#38; Technology Co. Ltd.</td>
			<td>Tanon- 
			2500</td>
			<td>an equipment for visualization of DNA band on an agarose gel or flavonoid spot on a polyamide TLC plate</td>
		</tr>
		<tr>
			<td>GenElute Plasmid Miniprep Kit</td>
			<td>Sigma-Aldrich Co. LLC</td>
			<td>PLN350-1KT</td>
			<td>minipreparation of plasmids</td>
		</tr>
		<tr>
			<td>kaempferol</td>
			<td>Sigma-Aldrich Co. LLC</td>
			<td>60010</td>
			<td>final reaction product and standard substance</td>
		</tr>
		<tr>
			<td>MassHunter Quanlitative Analysis (version B.01.04)</td>
			<td>Agilent Technologies, Inc</td>
			<td>N/A</td>
			<td>a software for analysis of HPLC/LC/MS data</td>
		</tr>
		<tr>
			<td>NanoDrop Microvolume UV-Vis Spectrophotometer</td>
			<td>Thermo Fisher Scientific</td>
			<td>ND-8000-GL</td>
			<td>an equipment for determination of DNA/RNA concentration</td>
		</tr>
		<tr>
			<td>naringenin</td>
			<td>Sigma-Aldrich Co. LLC</td>
			<td>N5893</td>
			<td>substrate for producing kaempferol</td>
		</tr>
		<tr>
			<td>Ni-IDA Agarose Resin</td>
			<td>Beijing CoWin Biotech Co., Ltd</td>
			<td>CW0010</td>
			<td>purification of His-tagged fusion proteins</td>
		</tr>
		<tr>
			<td>pET-32a(+)</td>
			<td>Novagen</td>
			<td>69015-3</td>
			<td>plasmid for cloning and expressing target genes</td>
		</tr>
		<tr>
			<td>plasmid sequencing</td>
			<td>GENEWIZ Suzhou</td>
			<td>N/A</td>
			<td>sequencing of recombinant plasmids</td>
		</tr>
		<tr>
			<td>primer synthesis</td>
			<td>GENEWIZ Suzhou</td>
			<td>N/A</td>
			<td>synthesis of PCR primers</td>
		</tr>
		<tr>
			<td>quercetin</td>
			<td>Shanghai Aladdin Biochemical Technology Co.,Ltd.</td>
			<td>Q111273</td>
			<td>final reaction product and standard substance</td>
		</tr>
		<tr>
			<td>SuperRT cDNA Synthesis Kit</td>
			<td>Beijing CoWin Biotech Co., Ltd</td>
			<td>CW0741</td>
			<td>synthesis of the first strand of cDNA from total RNA</td>
		</tr>
		<tr>
			<td>T4 DNA Ligase</td>
			<td>Thermo Fisher Scientific</td>
			<td>EL0016</td>
			<td>ligation of an insert into a linearized vector DNA</td>
		</tr>
		<tr>
			<td>Trizol</td>
			<td>Thermo Fisher Scientific</td>
			<td>15596018</td>
			<td>isolation of total RNA</td>
		</tr>
		<tr>
			<td>Vector NTI Advance</td>
			<td>Thermo Fisher Scientific</td>
			<td>12605099</td>
			<td>a software for PCR primer design and DNA sequence analysis</td>
		</tr>
		<tr>
			<td>Xcalibur v2.0.7</td>
			<td>Thermo Fisher Scientific</td>
			<td>N/A</td>
			<td>a software for analysis of HPLC data</td>
		</tr>
	</tbody>
</table>

biosynthesis of a flavonol from a flavanone by establishing a one-pot bienzymatic cascade

黄酮类是黄酮类的主要亚类,具有多种生物和药理活性。在这里,我们提供了一种体外酶合成黄酮醇的方法。在这种方法中,Atf3h和Atfls1是黄酮醇生物合成途径中的两个关键基因,在大肠杆菌中被克隆和过度表达。重组酶通过亲和柱进行纯化,然后在特定的合成缓冲液中建立双酶级联。本系统中合成了两种黄酮醇,并通过TLC和HPLC/LC/MS分析确定。该方法在黄酮醇的衍生中表现出明显的优势。既省时又省力,而且极具成本效益。反应易于精确控制,从而达到批量生产的规模。由于系统中的组件简单,目标产品易于纯化。然而,这个系统通常仅限于生产黄酮黄酮黄酮。

F3H和FLS1是植物中黄酮转化为黄酮醇的两种重要关键酶,如图1所示。开发一种体外生物合成系统,用于从黄酮Atf3h(GenBank加入号)中生产黄酮醇。NM_114983.3)和Atfls1(根银行加入号NM_120951.3)基因从4周大的A.thaliana幼苗中克隆成原核表达载体pET-32a(+)。重组质粒转化为大肠杆菌BL21(DE3),在IPTG诱导后表达融合蛋白,然后用Ni-IDA抗氧化树脂进行纯化。...

Watch this Scientific Journal Video about Biosynthesis of a Flavonol from a Flavanone by Establishing a One-pot Bienzymatic Cascade at JoVE.com

Biosynthesis of a Flavonol from a Flavanone by Establishing a One-pot Bienzymatic Cascade

黄酮醇的衍生对于其在医疗保健和食品工业中的应用至关重要。在这里,我们为黄酮中黄酮的生物合成提供详细的方案,并讨论与其他方法不同的关键步骤及其优势。

通过建立单罐双酶级联,从弗拉瓦内生物合成一种黄酮

Flavonols are a major subclass of flavonoids with a variety of biological and pharmacological activities. Here, we provide a method for the in vitro ...

使用该协议，我们可以轻松地在一个锅中从黄酮中产生大量的黄酮。这种技术省时、省、省、地高、易于控制，具有巨大的产业化潜力。是的，此方法可以提供其他二级代谢物的经济生产见解，但应用到它时需要一些修改。他通常一无所获或产量很低。我的建议是处理冰上重组酶，并在酶的库存溶液中加入10%甘油。这是因为只有通过视觉演示，新手才能了解影响实验成功实施的重要细节和真相。首先准备缓冲区。通过将适当量的TRIS基、抗坏血酸钠、α酮糖酸溶解到去离子水中，将适当量的甘油移液器溶解到溶液中，使合成缓冲液得到两倍。使用移液器添加盐酸将 pH 调节到 7.2，并通过添加去离子水来调节体积。将两次合成缓冲液存放在摄氏四度，供将来使用。然后在去硫化水中溶解硫酸铁六合液，使库存溶液得到100倍。在甲醇中溶解黄酮类化合物，使25毫摩尔黄酮的库存溶液，并在零下20摄氏度储存溶液。最关键的一步是建立一个合成系统。要建立一个合成系统，以生产黄酮中的黄酮，首先根据本表中列出的试剂在两毫升管中准备合成系统。将打开的管子在 40 摄氏度的摇晃热块中，在 600 rpm 下放置 40 分钟。之后，加入10微升醋酸和100微升乙酸乙酸乙酯，终止反应。两小时后，使用移液器将顶部有机相转至1.5毫升管，并在室温下将管放在烟罩中进行空气干燥。要执行TLC分析，首先从烟罩中取回管子，并在管中重新溶解160微升甲醇中的黄酮粉。通过稀释甲醇中的25毫摩尔类黄酮库存溶液，准备具有序列浓度的正宗黄酮样品。将重新溶解反应样品的一微升和正宗黄酮样品的一微升加载到聚酰胺板上。对同一板上其他浓度的正宗黄酮进行同样的测量。然后在烟气罩中，将样品加载板放入充满溶剂系统的色谱瓶中。将板连接到气缸底部，并在溶剂系统中运行板 20 分钟。空气干燥板在室温下。使用喷雾瓶，用1%乙醇溶液氯化铝喷洒板，然后再次在室温下空气干燥30分钟。在 254 纳米的紫外光下可视化板上的斑点，然后拍摄图像，然后在计算机上打开软件 ImageJ。单击文件，打开，打开要分析的图像。单击 ImageJ 用户界面中最矩形的选择工具。用鼠标勾勒出图像中的 ROI，然后按一个鼠标标记第一个 ROI。将鼠标右侧的矩形选择移到下一个 ROI，然后按两个键标记第二个 ROI。重复重复按 2 来标记所有其他 RBI。按三个在弹出窗口中为所有 RBI 生成配置文件图。然后，使用直线选择工具放置基线，以便为每个感兴趣的峰值定义一个封闭区域。通过单击 ImageJ 用户界面中的相应图标来激活魔杖工具。单击峰值内可显示弹出窗口中所有峰值的结果。接下来，在Excel中，根据相应的黄酮类化合物浓度绘制弹出窗口中的灰色值，以绘制真实黄酮的基于TLC的标准曲线。然后根据生成的公式计算本协议中产生的类黄酮的收益率。使用注射器通过 0.45 微米和 0.22 微米过滤器按顺序处理每个样品。通过吸油喷嘴将样品加载到 HPLC-MS 系统中，并使用 C18 柱在 30 摄氏度下分离样品。以每分钟一毫升的梯度将柱水，以10至85体积的乙酰硝酸在水中，并监测200至800纳米的亚硝酸盐的吸光。在负离子模式下执行 LC-MS 分析，在 300 摄氏度下以每分钟 10 升的干燥氮流量，在 250 摄氏度下以每分钟 7 升的护套气体流进行干燥氮流量，并使用内置软件收集数据。要提取单波长色谱仪，请打开定性分析程序，然后单击文件，打开数据文件。选择要在打开的数据文件窗口中分析的文件，然后单击"打开"以打开文件。右键单击色谱图结果窗口中的鼠标，然后在弹出式菜单中提取色谱图。在类型列表中，单击其他色谱图。在探测器组合框中，选择 DAD1，然后单击"确定"以在色谱图结果窗口中显示 HPLC 结果。单击停靠在色谱图结果窗口顶部的手动集成图标。为鼠标手动集成分析所需的峰值绘制基线。单击视图，集成峰值列表，以显示结果。将峰值区域的结果复制到 Excel，根据相应的黄酮类化合物浓度绘制基于 HPLC 的正宗黄酮标准曲线，然后根据生成的公式计算本协议中产生的类黄酮的收益率。然后重复相同的过程，提取单波长色谱图，以分析MS数据，以准确质量的黄酮类化合物。单击色谱图结果工具栏上的范围选择图标。选择兴趣的峰值。右键单击选定范围内的鼠标，然后单击弹出式菜单中的提取 MS 频谱，以在 MS 频谱结果窗口中显示结果。为了确定此体外系统是否可用于将黄酮转换为黄酮，将 NRN 添加到系统中。聚酰胺TLC板上出现了两个新点。一个点显示迁移距离与DHK相似，其他地点与KMF相似。HPLC 和 LC-MS 的进一步分析表明，新化学品在 Z 的准分子离子峰值 M 中分别表现出 12 分钟和 20 分钟的保留时间，与 DHK 和 KMF 相同。为了确定系统是否可用于将其他黄酮转化为相应的黄酮，ERD 被添加到系统中。聚酰胺TLC板上的两个新点分别显示了与DHQ和QRC相似的迁移距离。HPLC 和 LC-MS 分析表明，这些新化学品的保留时间分别为 10 分钟和 16 分钟，在 Z 上具有 303 和 301 的准分子离子峰值 M，与 DHQ 和 QRC 的保留时间完全一样。在准备合成系统时，应最后添加酶，并添加硫酸铁第二至最后。是的，它为其他二级代谢物的经济生产提供了指南。

Research

JoVE Journal

Biochemistry

A Tailored HPLC Purification Protocol That Yields High-purity Amyloid Beta 42 and Amyloid Beta 40 Peptides, Capable of Oligomer Formation

一个定制HPLC纯化协议，产量高纯度淀粉样蛋白β42和贝塔淀粉样蛋白肽40，有能力寡聚物形成的

Amyloidogenic peptides such as the Alzheimer's disease-implicated Amyloid beta (A&#946;), can present a significant challenge when trying to obtain high ...

科研

生物化学

Measuring Protein Binding to F-actin by Co-sedimentation

通过共沉淀测量蛋白与F-肌动蛋白的结合

Filamentous actin (F-actin) organization within cells is regulated by a large number of actin-binding proteins that control actin nucleation, growth, ...

Hydrolysis of a Ni-Schiff-Base Complex Using Conditions Suitable for Retention of Acid-labile Protecting Groups

镍 - 席夫碱的水解复杂环境条件适宜酸不稳定保护基团的保留

Unnatural amino acids, amino acids containing side-chain functionalities not commonly seen in nature, are increasingly found in synthetic peptide ...

Protein Film Infrared Electrochemistry Demonstrated for Study of H2 Oxidation by a [NiFe] Hydrogenase

Protein Film Infrared Electrochemistry Demonstrated for Study of H2 Oxidation by a [NiFe] Hydrogenase

用 [NiFe] 氢对 H2氧化的研究表明, 蛋白质膜红外电化学

Understanding the chemistry of redox proteins demands methods that provide precise control over redox centers within the protein. The technique of protein ...

A Yeast 2-Hybrid Screen in Batch to Compare Protein Interactions

酵母 2-混合筛在批次中比较蛋白质相互作用

Screening for protein-protein interactions using the yeast 2-hybrid assay has long been an effective tool, but its use has largely been limited to the ...

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

通过肽浓缩和质谱检测蛋白质泛化位点检测

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ...

Isolation of Lipoprotein Particles from Chicken Egg Yolk for the Study of Bacterial Pathogen Fatty Acid Incorporation into Membrane Phospholipids

从鸡蛋黄中分离脂蛋白颗粒,研究细菌病原体脂肪酸并入膜磷脂

Staphylococcus aureus and other Gram-positive pathogens incorporate fatty acids from the environment into membrane phospholipids. During infection, the ...

Detergent-assisted Reconstitution of Recombinant Drosophila Atlastin into Liposomes for Lipid-mixing Assays

Detergent-assisted Reconstitution of Recombinant Drosophila Atlastin into Liposomes for Lipid-mixing Assays

洗涤剂辅助重组重组果蝇阿拉他素到脂质混合测定脂质体

Membrane fusion is a crucial process in the eukaryotic cell. Specialized proteins are necessary to catalyze fusion. Atlastins are endoplasmic reticulum ...

Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH

单分子鱼的转录活性等位基因单细胞分析

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk ...

Removal and Replacement of Endogenous Ligands from Lipid-Bound Proteins and Allergens

从脂质绑定蛋白和过敏原中去除和替换内源性利根

Many major allergens bind to hydrophobic lipid-like molecules, including Mus m 1, Bet v 1, Der p 2, and Fel d 1. These ligands are strongly retained and ...