提交人感谢 NHLBI/nih (K08 HL124068 和 LRP HMOT1243)、NCATS/nih 通过 UCSF 临床和转化科学研究所催化剂方案 (UL1 TR000004)、UCSF 学术参议院、赫尔曼基金会提供的慷慨资助,基列科学研究学者奖, 辉瑞渴望心血管奖 (全部对 John s Chorba) 和霍华德休斯医学研究所 (阿德里 m. 盖文和凯万 m. Shokat)。

1. 蛋白酶载体的定点诱变
<ol><li>设计和订购自定义合成的寡核苷酸, 通过修改标准站点定向突变协议来安装感兴趣的变异12。标准淡化底漆 (没有额外的净化) 是完全可以接受的。 注: 对底漆设计的一般方法涉及创建部分重叠的底漆, 如表 1所示, 使用的熔融温度 (Tm) 计算器特定的聚合酶的兴趣。</li>
	<li>如表 2所示, 在冰上设置聚合酶链反应 (PCRs) 进行现场定向突变。</li>
	<li>按表 3所示循环 PCRs。</li>
	<li>运行 2-5 µL 的每一个 PCR 在1% 琼脂糖凝胶和可视化的产品, 以确认成功的放大。 注意: 一个成功的 PCR 将显示一个单一的, 突出的 DNA 波段在大约大小标记的模板 (~ 7.8 kB)。</li>
	<li>添加0.5 µL (每 25-µL 反应) 的 DpnI 和孵化样品37°c 1 小时。</li>
	<li>将反应的2µL 转化为具有化学 能力的大肠杆菌。 注: 快速生长的大肠杆菌菌株将减少潜伏期, 但任何克隆菌株都是可以接受的。</li>
	<li>将转换盘到含有 50-100 µg/毫升羧苄青霉素的 Luria-Bertani (磅) 琼脂上。在37摄氏度一夜之间孵化盘子。</li>
	<li>选择 2-4 个殖民地生长在一个小规模的文化 (2-5 毫升的 LB 培养基与 50-100 µg/毫升羧苄青霉素)。孵化的文化在37°c 在 220 rpm, 直到它是混浊的。</li>
	<li>根据制造商的指示, 用质粒 dna 纯化 (即"miniprep") 试剂盒将质粒 dna 从细胞中分离出来。用 microvolume 分光光度法定量测定洗脱 DNA 的浓度。</li>
	<li>用一个桑格测序服务 (如核心或商业设施) 广泛地序列质粒 DNA。根据服务规范准备 DNA 样品。在前测序反应中成功使用的底漆如表 4所述。 注: 由于整个质粒的 PCR 扩增, 建议每个突变体对整个 PCSK9 编码区域进行排序。</li>
</ol>2. 高通量荧光素酶的蛋白质水解测定方法
<ol><li>
细胞电镀 (0 天)
	<ol>
<li>使用低通道 HEK293T 细胞进行实验, 并在 Dulbecco 修饰的鹰培养基 (DMEM) 中培养10% 胎牛血清 (血清)。在组织培养罩中进行无菌条件下的所有细胞工作, 直到准备好化验荧光素酶活性。估计试验所需的水井和板材的数量, 预计 transfections 将在每一个条件下进行三个测试。</li>
		<li>将 HEK293T 细胞从母瓶中分离出来, 以最小体积的0.05% 胰蛋白酶-乙二胺-四乙酸 (EDTA) 来覆盖细胞。用2卷 DMEM 补充10% 的血清, 并将细胞转移到无菌管中, 使胰蛋白酶-EDTA 失效。使用自动单元格计数器计数单元格 (并用台盼蓝染色)。离心管在 500 x g 5 分钟恢复细胞。</li>
		<li>吸入含有胰蛋白酶的培养基, 并将培养基中的细胞重组为 2 x 105细胞/毫升的浓度。使用多通道吸管, 将100µL 的细胞转移到白色 (不透明的底部) 96 井板的每个井中, 从而使最终的播种浓度为 2 x 104细胞/井。 注: 对于最初的实验, 可能还会有帮助, 另外种子一个姐妹, 清底96井板, 以监测细胞生长和遵守在协议期间, 并指导未来的诊断。</li>
		<li>孵育细胞在37°c 和 5% CO2为 24 h。</li>
		<li>用96井格式准备一盘质粒的主板。将洗脱缓冲液中的每种质粒 (三 HCl、pH 值 8.5) 稀释成96井板的单个井中的50µL。<ol>
<li>为正控制质粒8、阴性对照 (S386A) 质粒8、无质粒缓冲 (模拟 transfections) 准备4口井。如果计划采用药物或其他细胞治疗方法, 则在每口井中用正控制质粒 (S386A) 和4口井分别制备主板, 每种均为阴性对照 () 质粒和无质粒缓冲液。</li>
		</ol>
</li>
	</ol>
</li>
	<li>
转染 (1 天)
	<ol>
<li>用深井96井板, 用96井板格式制备转染混合物。执行 transfections, 确保准备足够的试剂, 以解释吹打和转移损失。 注: 以下计算准备足够的试剂来运行一个96井板 (420 井保守估计)。<ol>
<li>将稀释后的脂质转染试剂混合, 将50.4 µL 试剂添加到2050µL 的降血清培养基中。制作一个 DNA precomplexation 试剂的总组合, 将33.6 µL 试剂添加到减少血清培养基的1730µL 中。</li>
			<li>使用多通道吸管, 整除16.8 µL 稀释的 DNA precomplexation 试剂混合物进入深井板块的每一井。</li>
			<li>使用多通道吸管, 整除3.2 µL (160 ng) 的每种质粒从主板进入深井板块的每一井。</li>
			<li>使用多通道吸管, 整除20µL 稀释脂转染试剂混合物的每一个良好的板块, 并混合的内容, 每个井使用多通道吸管。盖上盘子, 让他们坐在室温下 (RT) 10-15 分钟形成脂质: DNA 复合物。 注: 在转染后, 每井的最终成分将如下:40 的 dna, 0.12 µL 的转染试剂, 0.08 µL 的 dna 预络试剂, 每个井的含量将是10µL 在总容积 (减少血清培养基)。</li>
		</ol>
</li>
		<li>用多通道吸管轻轻地在96井板上的293T 细胞上交换培养基, 注意不要扰乱细胞。更换吸气介质与95µL DMEM 补充10% 的血清。 注意: 这将是一个适当的时间来治疗细胞与任何药物的利益。</li>
		<li>通过多通道吸管将转染混合物的10µL 添加到每个适当的井中。轻轻地旋转盘子, 混合在井中的内容。孵育板材在37°c 与 5% CO2为 24 h。 注: 油井的最终容积为105µL, 占24小时的蒸发量。</li>
	</ol>
</li>
	<li>
化验 (2 天)
	<ol>
<li>准备腔肠素试剂的库存溶液: 3 M 抗坏血酸钠 [溶于磷酸盐缓冲盐水 (PBS), 制备新鲜], 5 米氯化钠, 10 毫克/毫升牛血清白蛋白 (BSA; 在 PBS 中溶解, 准备新鲜) 和2毫米腔肠素 (溶于酸化甲醇, 含200µL 3 氮盐酸每10毫升)。 注: 2 毫米腔肠素可以储存2周, 当它保持在-80 摄氏度和没有光。</li>
		<li>根据表 5, 为荧光素酶的读数准备2x 腔肠素试剂, 每个细胞和培养基都有一个单独的试剂。混合所有试剂保存腔肠素首先, 过滤混合物通过一个0.22 µm 注射器过滤器, 然后添加腔肠素。保护试剂不受光, 直到它们准备好添加到盘子里。 注: 由于过滤器失去了溶液, 所以要做足够的试剂来说明过滤器的损耗以及转移的损失。表 5显示了2x 试剂的最终浓度 (以及添加到一个试剂的 96-井板中的库存溶液的数量)。</li>
		<li>在转染后从孵化箱中取出细胞24小时。使用多通道吸管, 把50µL 的条件介质从每个井转移到一个新鲜的, 白色底 (不透明) 的96井板。<ol>
<li>将车牌标记为它们是否包含介质或单元格。如果有超过一个96井板被转染, 标签的板块, 以确保每一个中载板与其母板的细胞。</li>
		</ol>
</li>
		<li>使用多通道吸管, 添加50µL 2x 非裂解腔肠素试剂的板只含有条件的培养基。在 5-10 分钟的 RT 中, 轻轻摇动或摇动盘子。</li>
		<li>使用多通道吸管, 添加50µL 的2x 裂解腔肠素试剂的板块包含细胞。在 5-10 分钟的 RT 中, 轻轻摇动或摇动盘子。</li>
		<li>孵化后, 读出平板阅读器中只中板的发光。然后, 阅读同一版阅读器中的含细胞板的发光。</li>
		<li>丢弃车牌并保存文件进行数据分析。</li>
	</ol>
</li>
</ol>3. 数据分析
<ol><li>使用电子表格软件执行初始数据分析。创建包含单元格和中板结果的电子表格。</li>
	<li>手动检查单元格中的数据以识别转染不良的油井。显示 < 5%-10% 的读数的负控制 (S386A) 质粒应该被认为是不良的转染, 使这些数据的解释可疑的水井。</li>
	<li>计算模拟转染井各板块的平均背景发光。从该板块的值中减去每个板块的背景。 注意: 这个值可能是微不足道的, 这取决于使用的板阅读器。</li>
	<li>通过计算荧光素酶活性在培养基中的比例, 与各井的总荧光素酶活性相比, 对数据进行处理。由于单元格板包含除细胞外的条件介质, 而中只板含有与细胞板相同数量的条件介质, 因此使用以下方程式是适当的: <img alt="Equation 1" src="/files/ftp_upload/58265/58265eq1.jpg"> 在这里, RLU = 相对发光单位, 背景减去读数从板读取器。</li>
	<li>计算阳性对照 (S386A) 和负控 (i.) 井的平均分泌荧光素酶。</li>
	<li>通过计算 Z 因子13来评估实验的整体质量: <img alt="Equation 2" src="/files/ftp_upload/58265/58265eq2.jpg"> 注意: 活动值来自正控制 (小波), 非活动值来自负控制 (S386A) 井。接近1的值表明实验质量较高。如果值低于 0, 请考虑重复该实验或优化工作流。</li>
	<li>将数据从电子表格程序传输到科学数据分析软件中。</li>
	<li>将分泌的荧光素酶活性规范化为阳性对照值和阴性对照 (S386A), 将阳性对照设置为 1, 负控制为0。</li>
	<li>使用回归和离群删除 (溃败) 方法清除异常值的数据, 将最大错误发现率设置为1%。</li>
	<li>通过将每个突变条件 (或突变体) 的数据与 "重量" 活动的平均值进行比较 (规范化为 1) 来评估统计学意义上的差异。使用 Sidak 法和α = 0.05 进行多个配对t检验, 修正多种比较。</li>
</ol>

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作者没有什么可透露的。

上述实验程序提出了一种克服单周转蛋白酶 PCSK9 内在低活性的方法, 并以稳健的方式评价其蛋白水解功能。该检测的关键概念依赖于将单个周转事件转换为酶放大读数。该检测的优点包括相对较短的时间框架和易用性荧光素酶的记者, 以及它的可伸缩性高吞吐量的方法。此外, 该化验结果评估蛋白质水解在其本机, 细胞的上下文。此外, 通过这种检测, 临床鉴定的 snp 可以评估其对 PCSK9 蛋白水解的影...

PCSK9 靶向 ldl 受体 (ldl R) 降解, 提高 ldl 胆固醇 (ldl C) 和驱动动脉粥样硬化性心脏病1,2。治疗靶向 PCSK9 稳健低密度脂蛋白 C 和改善心血管结局的患者, 即使添加到积极的降脂治疗与他汀类药物3,4。然而, 目前批准的疗法仅限于基于抗体的方法, 而且缺乏成本效益5,6。为了解决这个问题, 不那么昂贵的治疗方案, 一种方法来识别患者可能获得更大的利益, 或两者, 是需要的。
小分子方法可以针对细胞内 PCSK9, 提供一个改进的管理路线, 并降低成本, 使它们成为这个领域的 "圣杯"7。然而, PCSK9 已经被小分子所证明是难以用药的。作为蛋白酶, 靶向 PCSK9's 蛋白水解功能是一个很有吸引力的策略, 因为自我分解是 PCSK9 成熟8的速率限制步骤, 它对 LDL-R9的最大影响是必需的。然而, 到目前为止, 这项战略还没有成功, 可能是由于 PCSK9's 独特的生物化学: PCSK9 仅10, 执行单翻转反应, 自解裂后, PCSK9 prodomain 仍然绑定在活动站点作为自动抑制剂11, 防止任何进一步的蛋白酶活动的读数。
本文提出了一种评价高通量时尚8PCSK9 蛋白水解功能的方法。通过现场定向诱变, 研究者可以利用这一方法来探讨在临床中发现的编码 snp 的影响, 以评估它们对蛋白质水解的影响, PCSK9 成熟的速率限制步骤。此外, 这种方法将有助于设计高通量的屏幕, 以确定 PCSK9 蛋白降解的调制器, 这将最终扰乱 PCSK9 的表现为 LDL R (和调制 PCSK9's 胆固醇效应).最后, 该协议可以适应其他具有本质上低活性的蛋白酶, 只要 i) 一个特定的基质蛋白酶对可以找到, 和 ii) 一个合适的细胞内锚可以建立的基质。

<table>
	<tbody>
		<tr>
			<td>PCR Tubes</td>
			<td>USA Scientific</td>
			<td>1402-2900</td>
			<td>For PCR</td>
		</tr>
		<tr>
			<td>Q5 Hot Start</td>
			<td>New England Biolabs</td>
			<td>M0493L</td>
			<td>High-fidelity DNA Polymerase</td>
		</tr>
		<tr>
			<td>Deoxynucleotide Solution Mix</td>
			<td>New England Biolabs</td>
			<td>N0447L</td>
			<td>dNTPs (for PCR)</td>
		</tr>
		<tr>
			<td>pPCSK9-NLucProteaseAssay-WT</td>
			<td>Authors</td>
			<td>n/a</td>
			<td>Available from authors</td>
		</tr>
		<tr>
			<td>pPCSK9-NLucProteaseAssay-S386A</td>
			<td>Authors</td>
			<td>n/a</td>
			<td>Available from authors</td>
		</tr>
		<tr>
			<td>Agarose LE</td>
			<td>Gold Biotechnology</td>
			<td>A-201-100</td>
			<td>For DNA gels</td>
		</tr>
		<tr>
			<td>E-Gel Imager System with Blue Light Base</td>
			<td>ThermoFisher Scientific</td>
			<td>4466612</td>
			<td>For imaging DNA gels</td>
		</tr>
		<tr>
			<td>SYBR Safe DNA Gel Stain</td>
			<td>ThermoFisher Scientific</td>
			<td>S33102</td>
			<td>For DNA gels</td>
		</tr>
		<tr>
			<td>Tris Base</td>
			<td>ThermoFisher Scientific</td>
			<td>BP152-1</td>
			<td>For DNA gel running buffer</td>
		</tr>
		<tr>
			<td>Glacial acetic acid</td>
			<td>ThermoFisher Scientific</td>
			<td>A38-500</td>
			<td>For DNA gel running buffer</td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid solution</td>
			<td>Millipore Sigma</td>
			<td>3690</td>
			<td>EDTA, for DNA gel running buffer</td>
		</tr>
		<tr>
			<td>1 kb DNA ladder</td>
			<td>Gold Biotechnology</td>
			<td>D010</td>
			<td>DNA ladder</td>
		</tr>
		<tr>
			<td>DpnI</td>
			<td>New England Biolabs</td>
			<td>R0176S</td>
			<td>Restriction enzyme</td>
		</tr>
		<tr>
			<td>LB Agar plates with 100 &#181;g/mL carbenicillin</td>
			<td>Teknova</td>
			<td>L1010</td>
			<td>LB-Carb plates</td>
		</tr>
		<tr>
			<td>One Shot Mach1 T Phage-Resistent Chemically Competent E. coli</td>
			<td>ThermoFisher Scientific</td>
			<td>C862003</td>
			<td>Chemically competent cells</td>
		</tr>
		<tr>
			<td>LB Broth, Miller</td>
			<td>ThermoFisher Scientific</td>
			<td>BP1426-2</td>
			<td>LB</td>
		</tr>
		<tr>
			<td>Carbenicillin</td>
			<td>Gold Biotechnology</td>
			<td>C-103-5</td>
			<td>Selective antibiotic</td>
		</tr>
		<tr>
			<td>E.Z.N.A. Plasmid Mini Kit I</td>
			<td>Omega BioTek</td>
			<td>D6942-02</td>
			<td>DNA Purification Miniprep kit</td>
		</tr>
		<tr>
			<td>NanoDrop 2000 Spectrophotomer</td>
			<td>ThermoFisher Scientific</td>
			<td>ND-2000C</td>
			<td>Spectrophotometer</td>
		</tr>
		<tr>
			<td>293T Cells</td>
			<td>American Tissue Culture Collection (ATCC)</td>
			<td>CRL-3216</td>
			<td>HEK 293T cells</td>
		</tr>
		<tr>
			<td>DMEM, high glucose, pyruvate</td>
			<td>ThermoFisher Scientific</td>
			<td>11995065</td>
			<td>DMEM, mammalian cell media</td>
		</tr>
		<tr>
			<td>Fetal Bovine Sera</td>
			<td>Axenia Biologix</td>
			<td>F001</td>
			<td>FBS</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.05%), phenol red</td>
			<td>ThermoFisher Scientific</td>
			<td>25300062</td>
			<td>Trypsin, for cell dissociation</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>ThermoFisher Scientific</td>
			<td>10010023</td>
			<td>PBS</td>
		</tr>
		<tr>
			<td>Countess automated cell counter</td>
			<td>ThermoFisher Scientific</td>
			<td>C10227</td>
			<td>Automated cell counting</td>
		</tr>
		<tr>
			<td>Countess cell counting chamber slides</td>
			<td>ThermoFisher Scientific</td>
			<td>C10228</td>
			<td>Slides for cell counting</td>
		</tr>
		<tr>
			<td>CELLSTAR Tissue Culture Plates, White, White-Bottom, with Lid</td>
			<td>Grenier Bio-One</td>
			<td>655083</td>
			<td>White, white-bottom 96 well plate</td>
		</tr>
		<tr>
			<td>TempPlate non-skirted 96-well PCR plate, natural</td>
			<td>USA Scientific</td>
			<td>1402-9596</td>
			<td>96 well plate for master plasmid plate</td>
		</tr>
		<tr>
			<td>Nunc 2.0mL DeepWell Plates</td>
			<td>ThermoFisher Scientific</td>
			<td>278743</td>
			<td>96 well deep well plate</td>
		</tr>
		<tr>
			<td>Lipofectamine 3000</td>
			<td>ThermoFisher Scientific</td>
			<td>L3000008</td>
			<td>Lipid transfection reagent, Lf3K</td>
		</tr>
		<tr>
			<td>P3000 Reagent</td>
			<td>ThermoFisher Scientific</td>
			<td>L3000008</td>
			<td>DNA pre-complexation reagent, provided with Lf3K</td>
		</tr>
		<tr>
			<td>OptiMEM I Reduced Serum Medium</td>
			<td>ThermoFisher Scientific</td>
			<td>31985062</td>
			<td>Reduced serum medium for transfection</td>
		</tr>
		<tr>
			<td>(+)-Sodium L-ascorbate</td>
			<td>Millipore Sigma</td>
			<td>A4034</td>
			<td>Sodium ascorbate</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Millipore Sigma</td>
			<td>S9888</td>
			<td>NaCl</td>
		</tr>
		<tr>
			<td>Albumin, Bovine Serum, Fraction V, Low Heavy Metals</td>
			<td>Millipore Sigma</td>
			<td>12659</td>
			<td>BSA</td>
		</tr>
		<tr>
			<td>Methanol (HPLC)</td>
			<td>ThermoFisher Scientific</td>
			<td>A4524</td>
			<td>MeOH</td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>VWR</td>
			<td>JT9535-2</td>
			<td>Concentrated HCl</td>
		</tr>
		<tr>
			<td>Coelenterazine</td>
			<td>Gold Biotechnology</td>
			<td>CZ2.5</td>
			<td>Luciferase substrate</td>
		</tr>
		<tr>
			<td>Syringe Filter, Sterile</td>
			<td>ThermoFisher Scientific</td>
			<td>09-720-3</td>
			<td>Sterile filter, PVDF, 0.22 &#181;m pore</td>
		</tr>
		<tr>
			<td>Pipet-Lite Multi Pipette L12-200XLS+</td>
			<td>Rainin</td>
			<td>17013810</td>
			<td>Multichannel pipette</td>
		</tr>
		<tr>
			<td>Pipet-Lite Multi Pipette L12-20XLS+</td>
			<td>Rainin</td>
			<td>17013808</td>
			<td>Multichannel pipette</td>
		</tr>
		<tr>
			<td>Pipet-Lite Multi Pipette L12-10XLS+</td>
			<td>Rainin</td>
			<td>17013807</td>
			<td>Multichannel pipette</td>
		</tr>
		<tr>
			<td>Reagent reservoir</td>
			<td>Corning</td>
			<td>4870</td>
			<td>Trough for reagents</td>
		</tr>
		<tr>
			<td>Centrifuge tubes, 15 mL</td>
			<td>ThermoFisher Scientific</td>
			<td>05-539-12</td>
			<td>15 mL tubes</td>
		</tr>
		<tr>
			<td>Centrifuge tubes, 50 mL</td>
			<td>Corning</td>
			<td>430829</td>
			<td>50 mL tubes</td>
		</tr>
		<tr>
			<td>Spark Microplate Reader</td>
			<td>Tecan</td>
			<td>N/a</td>
			<td>Plate Reader</td>
		</tr>
		<tr>
			<td>Excel</td>
			<td>Microsoft</td>
			<td>2016 for Mac</td>
			<td>Spreadsheet software</td>
		</tr>
		<tr>
			<td>Prism</td>
			<td>GraphPad Software</td>
			<td>v7</td>
			<td>Scientific data analysis software</td>
		</tr>
	</tbody>
</table>

a high-throughput luciferase assay to evaluate proteolysis of the single-turnover protease pcsk9

Proprotein convertase 枯草杆菌9型 (PCSK9) 是一种单周转蛋白酶, 它调节血清低密度脂蛋白 (LDL) 水平, 从而导致心血管疾病。尽管 PCSK9 蛋白水解是其完全胆固醇效果所必需的, 但对其蛋白水解功能的评价是有挑战性的: PCSK9 只知道它自己, 只经历一个单一的营业额, 并在蛋白质水解后, 保留其基底在其活性部位为自动抑制剂。这里提出的方法描述了一种克服这些挑战的试验。该方法的研究重点是细胞间蛋白水解, 并将成功的裂解与分泌的荧光素酶活性联系在一起, 可以很容易地在条件培养基中读出。通过突变、瞬变转染和荧光素酶读数的顺序步骤, 该方法可以在基因或分子摄动条件下, 在高通量的情况下 PCSK9 蛋白质水解。该系统非常适合于临床发现的无义单核苷酸多态性 (SNPs) 的生物化学评价, 以及筛选 PCSK9 蛋白水解物的小分子抑制剂。

高通量的蛋白质分解测定方法依赖于克服三大挑战。首先, 为了克服单周转 PCSK9 蛋白酶固有的低产量, PCSK9 蛋白酶缺乏抑制 prodomain, 与分裂序列的 prodomain 链接到一个荧光素酶, 可以分泌14。第二, 为了满足蛋白酶在复合体中的折叠与抑制 prodomain 的需要, 这两种多肽在细胞 coexpressed15,16,通过一个 bicistronic ?...

Watch this Scientific Journal Video about A High-Throughput Luciferase Assay to Evaluate Proteolysis of the Single-Turnover Protease PCSK9 at JoVE.com

A High-Throughput Luciferase Assay to Evaluate Proteolysis of the Single-Turnover Protease PCSK9

高通量荧光素酶法评价单周转蛋白酶 PCSK9 的蛋白质水解

本协议提出了一种评估细胞内一种内在的低活性、单周转蛋白酶的蛋白水解活性的方法。具体来说, 该方法用于评价 PCSK9 的蛋白水解活性, 这是脂代谢的关键驱动力, 其蛋白水解活性是其最终胆固醇功能所必需的。

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a single-turnover protease which regulates serum low-density lipoprotein (LDL) levels and, ...

a-high-throughput-luciferase-assay-to-evaluate-proteolysis-single

Research

JoVE Journal

Biochemistry

8.0K 次观看.  Zuckerberg San Francisco General and University of California San Francisco. 本协议提出了一种评估细胞内一种内在的低活性、单周转蛋白酶的蛋白水解活性的方法。具体来说, 该方法用于评价 PCSK9 的蛋白水解活性, 这是脂代谢的关键驱动力, 其蛋白水解活性是其最终胆固醇功能所必需的。

视频: A High-Throughput Luciferase Assay to Evaluate Proteolysis of the Single-Turnover Protease PCSK9

High-throughput Screening of Carbohydrate-degrading Enzymes Using Novel Insoluble Chromogenic Substrate Assay Kits

Carbohydrates active enzymes (CAZymes) have multiple roles in vivo and are widely used for industrial processing in the biofuel, textile, detergent, paper and food industries. A deeper understanding of CAZymes is important from both fundamental biology and industrial standpoints. Vast numbers of CAZymes exist in nature (especially in microorganisms) and hundreds of thousands have been cataloged and described in the carbohydrate active enzyme database (CAZy). However, the rate of discovery of putative enzymes has outstripped our ability to biochemically characterize their activities. One reason for this is that advances in genome and transcriptome sequencing, together with associated bioinformatics tools allow for rapid identification of candidate CAZymes, but technology for determining an enzyme's biochemical characteristics has advanced more slowly. To address this technology gap, a novel high-throughput assay kit based on insoluble chromogenic substrates is described here. Two distinct substrate types were produced: Chromogenic Polymer Hydrogel (CPH) substrates (made from purified polysaccharides and proteins) and Insoluble Chromogenic Biomass (ICB) substrates (made from complex biomass materials). Both CPH and ICB substrates are provided in a 96-well high-throughput assay system. The CPH substrates can be made in four different colors, enabling them to be mixed together and thus increasing assay throughput. The protocol describes a 96-well plate assay and illustrates how this assay can be used for screening the activities of enzymes, enzyme cocktails, and broths.

A high-throughput assay for enzyme screening is described. This multiplexed ready-to-use assay kit comprises of pre-chosen Chromogenic Polymer Hydrogel (CPH) substrates and complex Insoluble Chromogenic Biomass (ICB) substrates. Target enzymes are polysaccharide degrading endo-enzymes and proteases.

使用新型不溶性生色底物检测试剂盒碳水化合物降解酶的高通量筛选

Carbohydrates active enzymes (CAZymes) have multiple roles in vivo and are widely used for industrial processing in the biofuel, textile, detergent, paper ...

科研

生物化学

Method for Identifying Small Molecule Inhibitors of the Protein-protein Interaction Between HCN1 and TRIP8b

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the excitability of neurons. The subcellular distribution of these channels in pyramidal neurons of hippocampal area CA1 is regulated by tetratricopeptide repeat-containing Rab8b interacting protein (TRIP8b), an auxiliary subunit. Genetic knockout of HCN pore forming subunits or TRIP8b, both lead to an increase in antidepressant-like behavior, suggesting that limiting the function of HCN channels may be useful as a treatment for Major Depressive Disorder (MDD). Despite significant therapeutic interest, HCN channels are also expressed in the heart, where they regulate rhythmicity. To circumvent off-target issues associated with blocking cardiac HCN channels, our lab has recently proposed targeting the protein-protein interaction between HCN and TRIP8b in order to specifically disrupt HCN channel function in the brain. TRIP8b binds to HCN pore forming subunits at two distinct interaction sites, although here the focus is on the interaction between the tetratricopeptide repeat (TPR) domains of TRIP8b and the C terminal tail of HCN1. In this protocol, an expanded description of a method for purifying TRIP8b and executing a high throughput screen to identify small molecule inhibitors of the interaction between HCN and TRIP8b, is described. The method for high throughput screening utilizes a Fluorescence Polarization (FP) -based assay to monitor the binding of a large TRIP8b fragment to a fluorophore-tagged eleven amino acid peptide corresponding to the HCN1 C terminal tail. This method allows &#39;hit&#39; compounds to be identified based on the change in the polarization of emitted light. Validation assays are then performed to ensure that &#39;hit&#39; compounds are not artifactual.

The interaction between HCN channels and their auxiliary subunit has been identified as a therapeutic target in Major Depressive Disorder. Here, a fluorescence polarization-based method for identifying small molecule inhibitors of this protein-protein interaction, is presented.

方法确定HCN1和TRIP8b之间蛋白 - 蛋白相互作用的小分子抑制剂

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the ...

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages for identifying biologically active chemical structures that can modify complex phenotypes such as lifespan. Described here is a simple protocol for producing hundreds of 96-well culture plates with fairly consistent numbers of C. elegans in each well. Next, we specified how to use these cultures to screen thousands of chemicals for effects on the lifespan of the nematode C. elegans. This protocol makes use of temperature sensitive sterile strains, agar plate conditions, and simple animal handling to facilitate the rapid and high throughput production of synchronized animal cultures for screening.

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Here we describe a simple protocol for rapidly producing hundreds of nematode growth media agar, 96-well culture plates with consistent numbers of Caenorhhabditis elegans per well. These cultures are useful for the phenotypic screening of whole organisms. We focus here on using these cultures to screen chemicals for pro-longevity effects.

一种简单的高通量化学筛选方法在秀丽线虫中的应用

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages ...

Use of Recombinant Fusion Proteins in a Fluorescent Protease Assay Platform and Their In-gel Renaturation

Proteases are intensively studied enzymes due to their essential roles in several biological pathways of living organisms and in pathogenesis; therefore, they are important drug targets. We have developed a magnetic-agarose-bead-based assay platform for the investigation of proteolytic activity, which is based on the use of recombinant fusion protein substrates. In order to demonstrate the use of this assay system, a protocol is presented on the example of human immunodeficiency virus type 1 (HIV-1) protease. The introduced assay platform can be utilized efficiently in the biochemical characterization of proteases, including enzyme activity measurements in mutagenesis, kinetic, inhibition, or specificity studies, and it may be suitable for high-throughput substrate screening or may be adapted to other proteolytic enzymes.
In this assay system, the applied substrates contain N-terminal hexahistidine (His6) and maltose-binding protein (MBP) tags, cleavage sites for tobacco etch virus (TEV) and HIV-1 proteases, and a C-terminal fluorescent protein. The substrates can be efficiently produced in Escherichia coli cells and easily purified using nickel (Ni)-chelate-coated beads. During the assay, the proteolytic cleavage of bead-attached substrates leads to the release of fluorescent cleavage fragments, which can be measured by fluorimetry. Additionally, cleavage reactions can be analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A protocol for the in-gel renaturation of assay components is also described, as partial renaturation of fluorescent proteins enables their detection based on molecular weight and fluorescence.

Here, we present the detailed procedure of a recently developed protease assay platform utilizing N-terminal hexahistidine/maltose-binding protein and fluorescent protein-fused recombinant substrates attached to the surface of nickel-nitrilotriacetic acid magnetic agarose beads. A subsequent in-gel analysis of the assay samples separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is also presented.

重组融合蛋白在荧光蛋白酶检测平台中的应用及其凝胶内再生

Proteases are intensively studied enzymes due to their essential roles in several biological pathways of living organisms and in pathogenesis; therefore, ...

A Semi-High-Throughput Adaptation of the NADH-Coupled ATPase Assay for Screening Small Molecule Inhibitors

ATPase enzymes utilize the free energy stored in adenosine triphosphate to catalyze a wide variety of endergonic biochemical processes in vivo that would not occur spontaneously. These proteins are crucial for essentially all aspects of cellular life, including metabolism, cell division, responses to environmental changes and movement. The protocol presented here describes a nicotinamide adenine dinucleotide (NADH)-coupled ATPase assay that has been adapted to semi-high throughput screening of small molecule ATPase inhibitors. The assay has been applied to cardiac and skeletal muscle myosin II's, two actin-based molecular motor ATPases, as a proof of principle. The hydrolysis of ATP is coupled to the oxidation of NADH by enzymatic reactions in the assay. First, the ADP generated by the ATPase is regenerated to ATP by pyruvate kinase (PK). PK catalyzes the transition of phosphoenolpyruvate (PEP) to pyruvate in parallel. Subsequently, pyruvate is reduced to lactate by lactate dehydrogenase (LDH), which catalyzes the oxidation of NADH in parallel. Thus, the decrease in ATP concentration is directly correlated to the decrease in NADH concentration, which is followed by change to the intrinsic fluorescence of NADH. As long as PEP is available in the reaction system, the ADP concentration remains very low, avoiding inhibition of the ATPase enzyme by its own product. Moreover, the ATP concentration remains nearly constant, yielding linear time courses. The fluorescence is monitored continuously, which allows for easy estimation of the quality of data and helps to filter out potential artifacts (e.g., arising from compound precipitation or thermal changes).

A nicotinamide adenine dinucleotide (NADH)-coupled ATPase assay has been adapted to semihigh throughput screening of small molecule myosin inhibitors. This kinetic assay is run in a 384-well microplate format with total reaction volumes of only 20 &#181;L per well. The platform should be applicable to virtually any ADP producing enzyme.

用于筛选小分子抑制剂的NADH耦合ATPase分析的半高通量自适应

ATPase enzymes utilize the free energy stored in adenosine triphosphate to catalyze a wide variety of endergonic biochemical processes in vivo that would ...

High-Throughput Total Internal Reflection Fluorescence and Direct Stochastic Optical Reconstruction Microscopy Using a Photonic Chip

Total internal reflection fluorescence (TIRF) is commonly used in single molecule localization based super-resolution microscopy as it gives enhanced contrast due to optical sectioning. The conventional approach is to use high numerical aperture microscope TIRF objectives for both excitation and collection, severely limiting the field of view and throughput. We present a novel approach to generating TIRF excitation for imaging with optical waveguides, called chip-based nanoscopy. The aim of this protocol is to demonstrate how chip-based imaging is performed in an already built setup. The main advantage of chip-based nanoscopy is that the excitation and collection pathways are decoupled. Imaging can then be done with a low magnification lens, resulting in large field of view TIRF images, at the price of a small reduction in resolution. Liver sinusoidal endothelial cells (LSECs) were imaged using direct stochastic optical reconstruction microscopy (dSTORM), showing a resolution comparable to traditional super-resolution microscopes. In addition, we demonstrate the high-throughput capabilities by imaging a 500 &#181;m x 500 &#181;m region with a low magnification lens, providing a resolution of 76 nm. Through its compact character, chip-based imaging can be retrofitted into most common microscopes and can be combined with other on-chip optical techniques, such as on-chip sensing, spectroscopy, optical trapping, etc. The technique is thus ideally suited for high throughput 2D super-resolution imaging, but also offers great opportunities for multi-modal analysis.

Chip-based super-resolution optical microscopy is a novel approach to fluorescence microscopy and offers advantages in cost effectiveness and throughput. Here, the protocols for chip preparation and imaging are shown for TIRF microscopy and localization-based super-resolution microscopy.

使用光子芯片的高通量总内反射荧光和直接随机光学重建显微镜

Total internal reflection fluorescence (TIRF) is commonly used in single molecule localization based super-resolution microscopy as it gives enhanced ...

Click-Chemistry Based Fluorometric Assay for Apolipoprotein N-acyltransferase from Enzyme Characterization to High-Throughput Screening

Lipoproteins from proteobacteria are posttranslationally modified by fatty acids derived from membrane phospholipids by the action of three integral membrane enzymes, resulting in triacylated proteins. The first step in the lipoprotein modification pathway involves the transfer of a diacylglyceryl group from phosphatidylglycerol onto the prolipoprotein, resulting in diacylglyceryl prolipoprotein. In the second step, the signal peptide of prolipoprotein is cleaved, forming an apolipoprotein, which in turn is modified by a third fatty acid derived from a phospholipid. This last step is catalyzed by apolipoprotein N-acyltransferase (Lnt). The lipoprotein modification pathway is essential in most &#947;-proteobacteria, making it a potential target for the development of novel antibacterial agents. Described here is a sensitive assay for Lnt that is compatible with high-throughput screening of small inhibitory molecules. The enzyme and substrates are membrane-embedded molecules; therefore, the development of an in vitro test is not straightforward. This includes the purification of the active enzyme in the presence of detergent, the availability of alkyne-phospholipids and diacylglyceryl peptide substrates, and the reaction conditions in mixed micelles. Furthermore, in order to use the activity test in a high-throughput screening (HTS) setup, direct readout of the reaction product is preferred over coupled enzymatic reactions. In this fluorometric enzyme assay, the alkyne-triacylated peptide product is rendered fluorescent through a click-chemistry reaction and detected in a multiwell plate format. This method is applicable to other acyltransferases that use fatty acid-containing substrates, including phospholipids and acyl-CoA.

Presented here is a sensitive fluorescence assay to monitor apolipoprotein N-acyltransferase activity using diacylglyceryl peptide and alkyne-phospholipids as substrates with click-chemistry.

基于点击化学的载脂蛋白N-酰基转移酶荧光测定从酶表征到高通量筛选

Lipoproteins from proteobacteria are posttranslationally modified by fatty acids derived from membrane phospholipids by the action of three integral ...

A High-Throughput Enzyme-Coupled Activity Assay to Probe Small Molecule Interaction with the dNTPase SAMHD1

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) is a pivotal regulator of intracellular deoxynucleoside triphosphate (dNTP) pools, as this enzyme can hydrolyze dNTPs into their corresponding nucleosides and inorganic triphosphates. Due to its critical role in nucleotide metabolism, its association to several pathologies, and its role in therapy resistance, intense research is currently being carried out for a better understanding of both the regulation and cellular function of this enzyme. For this reason, development of simple and inexpensive high-throughput amenable methods to probe small molecule interaction with SAMHD1, such as allosteric regulators, substrates, or inhibitors, is vital. To this purpose, the enzyme-coupled malachite green assay is a simple and robust colorimetric assay that can be deployed in a 384-microwell plate format allowing the indirect measurement of SAMHD1 activity. As SAMHD1 releases the triphosphate group from nucleotide substrates, we can couple a pyrophosphatase activity to this reaction, thereby producing inorganic phosphate, which can be quantified by the malachite green reagent through the formation of a phosphomolybdate malachite green complex. Here, we show the application of this methodology to characterize known inhibitors of SAMHD1 and to decipher the mechanisms involved in SAMHD1 catalysis of non-canonical substrates and regulation by allosteric activators, exemplified by nucleoside-based anticancer drugs. Thus, the enzyme-coupled malachite green assay is a powerful tool to study SAMHD1, and furthermore, could also be utilized in the study of several enzymes which release phosphate species.

SAMHD1 is a deoxynucleoside triphosphate triphosphohydrolase with critical roles in human health and disease. Here we present a versatile enzyme-coupled SAMHD1 activity assay, deployed in a 384-well microplate format, that allows for the evaluation of small molecules and nucleotide analogues as SAMHD1 substrates, activators, and inhibitors.

一种高通量酶偶联活性测定法，用于探测小分子与 dNTP 酶 SAMHD1 的相互作用

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) is a pivotal regulator of intracellular deoxynucleoside triphosphate (dNTP) pools, as this ...

A "Dual-Addition" Calcium Fluorescence Assay for the High-Throughput Screening of Recombinant G Protein-Coupled Receptors

G protein-coupled receptors (GPCRs) represent the largest superfamily of receptors and are the targets of numerous human drugs. High-throughput screening (HTS) of random small molecule libraries against GPCRs is used by the pharmaceutical industry for target-specific drug discovery. In this study, an HTS was employed to identify novel small-molecule ligands of invertebrate-specific neuropeptide GPCRs as probes for physiological studies of vectors of deadly human and veterinary pathogens.
The invertebrate-specific kinin receptor was chosen as a target because it regulates many important physiological processes in invertebrates, including diuresis, feeding, and digestion. Furthermore, the pharmacology of many invertebrate GPCRs is poorly characterized or not characterized at all; therefore, the differential pharmacology of these groups of receptors with respect to the related GPCRs in other metazoans, especially humans, adds knowledge to the structure-activity relationships of GPCRs as a superfamily. An HTS assay was developed for cells in 384-well plates for the discovery of ligands of the kinin receptor from the cattle fever tick, or southern cattle tick, Rhipicephalus microplus. The tick kinin receptor was stably expressed in CHO-K1 cells.
The kinin receptor, when activated by endogenous kinin neuropeptides or other small molecule agonists, triggers Ca2+ release from calcium stores into the cytoplasm. This calcium fluorescence assay combined with a &#34;dual-addition&#34; approach can detect functional agonist and antagonist &#34;hit&#34; molecules in the same assay plate. Each assay was conducted using drug plates carrying an array of 320 random small molecules. A reliable Z&#39; factor of 0.7 was obtained, and three agonist and two antagonist hit molecules were identified when the HTS was at a 2 &#181;M final concentration. The calcium fluorescence assay reported here can be adapted to screen other GPCRs that activate the Ca2+ signaling cascade.

In this work, a high-throughput, intracellular calcium fluorescence assay for 384-well plates to screen small molecule libraries on recombinant G protein-coupled receptors (GPCRs) is described. The target, the kinin receptor from the cattle fever tick, Rhipicephalus microplus, is expressed in CHO-K1 cells. This assay identifies agonists and antagonists using the same cells in one "dual-addition" assay.

用于重组G蛋白偶联受体高通量筛选的"双加法"钙荧光测定

G protein-coupled receptors (GPCRs) represent the largest superfamily of receptors and are the targets of numerous human drugs. High-throughput screening ...

蛋白质结晶的高通量筛选

High-Throughput Screening to Obtain Crystal Hits for Protein Crystallography

X-ray crystallography is the most commonly employed technique to discern macromolecular structures, but the crucial step of crystallizing a protein into an ordered lattice amenable to diffraction remains challenging. The crystallization of biomolecules is largely experimentally defined, and this process can be labor-intensive and prohibitive to researchers at resource-limited institutions. At the National High-Throughput Crystallization (HTX) Center, highly reproducible methods have been implemented to facilitate crystal growth, including an automated high-throughput 1,536-well microbatch-under-oil plate setup designed to sample a wide breadth of crystallization parameters. Plates are monitored using state-of-the-art imaging modalities over the course of 6 weeks to provide insight into crystal growth, as well as to accurately distinguish valuable crystal hits. Furthermore, the implementation of a trained artificial intelligence scoring algorithm for identifying crystal hits, coupled with an open-source, user-friendly interface for viewing experimental images, streamlines the process of analyzing crystal growth images. Here, the key procedures and instrumentation are described for the preparation of the cocktails and crystallization plates, imaging the plates, and identifying hits in a way that ensures reproducibility and increases the likelihood of successful crystallization.

作者聚焦：高通量筛选以获得蛋白质晶体学的晶体命中  

This protocol details high-throughput crystallization screening, ranging from the 1,536 microassay plate preparation to the end of a 6 week experimental time window. Details are included about the sample setup, the imaging obtained, and how users can perform analyses using an artificial intelligence-enabled graphical user interface to quickly and efficiently identify macromolecular crystallization conditions.

高通量筛选以获得用于蛋白质晶体学的晶体命中

X-ray crystallography is the most commonly employed technique to discern macromolecular structures, but the crucial step of crystallizing a protein into ...