本出版物是根据国家科学基金会资助的。 车1708237

1. 净化准备
<ol><li>无细胞粗萃取物的制备<ol>
<li>获得一个9-10 克的大肠杆菌(BL21) 细胞颗粒, 抗原 polyhistidine 标记金属蛋白质25在50毫升锥管。</li>
		<li>添加5毫升每克室温裂解/捆绑缓冲 (50 毫米磷酸盐, 300 毫米氯化钠, 10 毫米咪唑在 pH 8) 到 ~ 9-10 g 的细胞丸为总容积 ~ 45-50 毫升。周期性涡刺激溶解。如果颗粒被冻结, 在温水浴中解冻。</li>
		<li>使用冰水浴, 使细胞悬浮液冷却至3摄氏度, 以制备细胞裂解。 注: 在这一点上, 细胞裂解的超声波, 珠铣或其他方法是可能的。珠铣是在这里展示, 因为它是快速, 相对便宜, 不需要耳保护。</li>
		<li>根据制造商的指示, 装配一个50毫升的珠铣削室。</li>
		<li>填充的珠磨室半满冷0.1 毫米玻璃珠, 已储存在-20 °c。</li>
		<li>用冷冻细胞悬浮液填满房间的其余部分, 并使用4°c 溶解/束缚缓冲器完全填满房间。</li>
		<li>用小刮刀旋转珠磨的轴, 并将电池悬浮与珠子混合。</li>
		<li>用吸管去掉任何大气泡, 然后拧紧盖子。</li>
		<li>从任何渗漏中把房间晾干。</li>
		<li>添加约1杯冰, 以清楚, 塑料夹克, 倒置填充50毫升室进入冰填充夹克, 和螺丝关闭。</li>
		<li>把冰套室固定在马达上。</li>
		<li>将珠磨马达打开15秒, 在 15800 rpm, 然后允许休息45秒。重复8次。</li>
		<li>当8循环完成, 醒酒整个细胞裂解悬浮成50毫升或更大的管额定高速离心 (2.5万 x 克或更高)。在离心机上放置一对平衡管, 在 4°c 2.5万 x g 处旋转40-45 分钟, 以颗粒细胞碎片和玻璃珠为单位。 注: 如果额定为高速离心的50毫升或更大的管子不可用, 则在50毫升圆锥管中, 用离心在 1000-2000 x g 处去除玻璃珠, 以产生体积较小的细胞悬浮液 (~ 25 毫升), 可排水成较小的体积e 管额定高速离心。</li>
		<li>当离心完成后, 醒酒清除, 黄色, 无细胞, 粗萃取物成一个干净的50毫升圆锥管。注意不要转移任何细胞碎片。</li>
		<li>收集一个小样本的无细胞的粗提取物, 以供以后分析的纯化由 SDS26页。</li>
	</ol>
</li>
	<li>IMAC 柱的制备<ol>
<li>获得一个 1.5 x 20 厘米列配备了较低的床支持, 以保留树脂颗粒, 鲁尔锁出口和上限, 也包含鲁尔锁配件。将鲁尔锁插座与旋塞阀配合使用以控制流量。</li>
		<li>在台式工作, 安全地将柱子放在环架上。</li>
		<li>在20% 乙醇中获得50% 的镍-nitriloacetic 酸 (镍-NTA) 树脂浆料, 其储存在4摄氏度。</li>
		<li>轻轻地旋转瓶子, 均匀地重新悬浮树脂。</li>
		<li>工作在室温和台式, 使用一个毕业的吸管提取2毫升的泥浆, 这将产生1毫升的固定树脂能够结合50-60 毫克的 polyhistidine 标签蛋白, 并将浆料转移到柱上。</li>
		<li>打开旋塞阀, 允许过剩的存储解决方案从树脂中排出重力。</li>
		<li>安全关闭旋塞阀</li>
		<li>使用巴斯德吸管, 小心地加入冷冻裂解/捆绑缓冲 (50 毫米磷酸盐, 300 毫米氯化钠, 10 毫米咪唑在 pH 8) 和到树脂柱-至少30毫升。注意不要扰乱树脂表面。在列的墙壁下运行缓冲区, 以防止飞溅。</li>
		<li>平衡树脂通过允许溶解/束缚缓冲慢慢地从专栏被重力排泄入收集烧杯。</li>
		<li>当裂解/捆绑缓冲液大部分耗尽时, 关闭旋塞阀以停止缓冲液的流动, 当5毫升的溶解缓冲液保持在树脂之上, 并离开柱, 直立, 直到准备进行或长达1周。</li>
	</ol>
</li>
</ol>2. IMAC 对 Polyhistidine 标记目标的纯化
<ol><li>准备洗涤缓冲器 (50 毫米磷酸盐, 300 毫米氯化钠, 20 毫米咪唑 pH 8) 和洗脱缓冲器 (50 毫米磷酸盐, 300 毫米氯化钠, 250 毫米咪唑, 10% 甘油 pH 值 8)。凉在4°c。</li>
	<li>通过打开旋塞阀, 并允许剩余的裂解/粘结缓冲液通过镍 NTA 树脂 NTA, 制备出无细胞粗萃取物的镍----------。当所有的缓冲液耗尽, 树脂表面暴露后, 关闭旋塞阀。</li>
	<li>使用巴斯德吸管, 小心地将无细胞的粗萃取物吸管到树脂上, 注意不要扰乱表面。在柱子的墙上运行粗糙的萃取物以防止溅水。所应用的粗提取物量取决于 polyhistidine 标记蛋白的表达水平;确保粗提取物的总体积包含50-60 毫克或更少的目标蛋白质每毫升镍 NTA 树脂 (见步骤 1.2.4)。</li>
	<li>当所有的粗萃取物都被转移到柱子上时, 打开旋塞阀, 这样柱子就会被重力排出。这种流动是由没有绑定到树脂的蛋白质组成的-收集 100 ul, 以供以后的分析纯化的 SDS 页。26
</li>
	<li>无细胞粗萃取物的粘度可以大大减慢柱体的重力流。要提高流量, 应用简单的 "手泵":<ol>
<li>采取一个10毫升鲁尔锁注射器, 并连接到柱帽上使用短长度的塑料油管和鲁尔锁配件之间的柱帽和注射器。拔下注射器柱塞, 然后将柱帽紧配合在柱子顶部。</li>
		<li>轻轻地压缩注射器的柱塞, 同时通过将淋洗收集到一个毕业的气缸来手动评估流速;每分钟1-2 毫升的速率与树脂和此设置兼容。当流速减慢时, 轻轻地增加注射器柱塞的压缩。</li>
		<li>为防止将空气引入树脂, 当应用液体的半月板达到5-10 毫米以上的树脂床, 并允许剩余的体积被重力排出时, 取下柱帽和附着的手泵。</li>
	</ol>
</li>
	<li>当无细胞的粗萃取物已完成排泄和树脂表面再次暴露, 使用巴斯德吸管小心地添加〜30毫升的冷冻洗涤缓冲柱-注意不要扰乱树脂表面。在列的墙壁下运行缓冲区, 以防止飞溅。</li>
	<li>打开旋塞阀, 并允许洗涤缓冲区在列中流失。这个过程是从树脂中去除弱束缚蛋白。丢弃淋洗。</li>
	<li>当洗涤缓冲器正在排水时, 准备十六, 标记, 离心管收集1毫升的分数。</li>
	<li>当洗涤缓冲液耗尽, 树脂表面再次暴露后, 关闭旋塞阀。使用巴斯德吸管小心地将30毫升的冷却洗脱缓冲液添加到柱上-注意不要扰乱树脂表面。在列的墙壁下运行缓冲区, 以防止飞溅。</li>
	<li>慢慢打开旋塞阀, 并允许洗脱缓冲洗脱 polyhistidine 标记的蛋白质从专栏。收集1毫升的淋洗在每个标记的离心管, 1 到16。</li>
	<li>根据试剂和/或仪器制造商27,28的具体方法, 测试蛋白质的分数, 如布拉德福德化验或紫外可见光光谱学。如图所示, 使用考马斯蓝的比色法可以缩小到 100 ul 体积, 以牺牲每一个分数的小体积。</li>
	<li>混合 3 ul 的每一个分数与100µL 的比色蛋白检测试剂, 并手动观察任何颜色的形成, 以表明存在蛋白质的分数;不需要用光谱仪进行检测。</li>
	<li>如果蛋白质在分数16中发现, 继续洗脱1毫升的分数, 并定期化验蛋白质, 直到洗脱分数不再含有大量的蛋白质。</li>
	<li>将含蛋白质的分数组合成一个干净的锥形管, 并提取 100 ul 样品, 供后期分析用 SDS26页。</li>
	<li>进行金属离子余子的重构, 或在整除数的3毫升内冷冻蛋白质。确保洗脱缓冲液中含有10% 甘油是一种在冷冻过程中稳定纯蛋白的 cryoprotectant。</li>
	<li>当 NTA 柱的洗脱是完整的, 即所有的蛋白质是从列和收集的分数, 传递另一个25毫升的洗脱缓冲通过该专栏, 并存储在4°c 5 毫升的洗脱缓冲, 以短期存储的列, 或裂解/捆绑缓冲器与20% 乙醇的长期储存。</li>
</ol>3. 用 Fe2 +重组靶蛋白
<ol><li>添加 Fe2 + 注: 非血红素铁 (II) 结合酶, 如 l-多巴酶在本例中, 需要一个 Fe2 +离子的活动;但是, 铁 (II) 容易氧化成铁 (III), 这是不活跃的, 和蛋白质的一部分, 没有任何铁净化。<ol>
<li>要开始重建金属, 获得3毫升的纯化酶悬浮在洗脱缓冲。如果结冰, 用温水浴迅速解冻, 一旦解冻, 将蛋白质放在冰上。</li>
		<li>利用试管中的蛋白质体积, 计算出所需的抗坏血酸钠 (198.1 克/摩尔), 使样品中的最终浓度为12.5 毫米。此外, 计算 dithiothreitol (154.25 克/摩尔) 所需的量, 使最终浓度12.5 毫米的样品。</li>
		<li>加入固体抗坏血酸钠, 并轻轻, 但彻底, 混合完全溶解。注意不要导致蛋白质沉淀过度积极的混合。添加少量的铁 (II) 硫酸水合物 (兆瓦278.01 克/摩尔) 的蛋白质样品。</li>
		<li>为了获得足够小的铁盐的数量, 轻拍1毫米颗粒的 FeSO4· 7H2O 固体在一张重纸上, 折叠纸在颗粒上, 并且粉碎它与金属铲的平的边。</li>
		<li>将产生的粉末添加到蛋白质管中。</li>
		<li>漩涡混合和玫瑰色粉红色的颜色将出现在管。</li>
		<li>在冰上孵化出蛋白质的粉红色溶液, 盖上10-30 分钟。粉红色的颜色会随着时间慢慢褪色。</li>
	</ol>
</li>
	<li>凝胶过滤<ol>
<li>使用10毫升球形聚丙烯酰胺凝胶填充的凝胶过滤柱, 其分子量排除限约为 6000, 在 1.5 x 12 厘米柱上90–180µm 的水合粒径范围, 该塔还配有10毫升储层。确保柱上有下部和上床支承, 其形式为30微米多孔聚乙烯圆盘, 保留树脂在柱上, 防止树脂床运行干燥。将凝胶过滤柱安装到环形支架上。</li>
		<li>如果使用预包装的凝胶过滤柱, 取下瓶盖, 并将多余的缓冲液倒在上床支架上方。</li>
		<li>要平衡柱, 首先用缓冲液与随后的化验和贮存相兼容的储层, 例如, 50 毫米 NaH2PO4, 200 毫米氯化钠, 10% 甘油在 pH 值8.0。</li>
		<li>如果使用预包装的凝胶过滤柱, 切断凝胶过滤塔的底部, 启动缓冲流, 并暴露鲁尔滑移管接头。将旋塞阀与列的鲁尔滑动端相匹配, 但保持打开状态, 并允许该列通过重力进行滴水。</li>
		<li>当缓冲区从柱体中排出, 上层支撑被暴露后, 关闭旋塞阀, 并将该溶液吹打到所暴露的上床支架上, 将金属酶重组后的 Fe (II) 的 ~ 3 毫升添加到柱上。</li>
		<li>打开旋塞阀, 允许整个3.0 毫升的样品通过重力进入柱。丢弃这些前3毫升的淋洗。在处理解决方案时形成的任何粉红色颜色都将被困在列的顶部。</li>
		<li>当柱停止滴水和上床支撑暴露, 添加4毫升的凝胶过滤缓冲 (例如, 50 毫米 NaH2PO4, 200 毫米氯化钠, 10% 甘油在 pH 8.0) 到专栏。打开旋塞阀, 并收集淋洗到离心管超过八0.5 毫升分数。</li>
		<li>用比前述的比色蛋白测定或紫外-27,28测定蛋白质的分数。</li>
		<li>结合含蛋白质的分数。</li>
		<li>提取26的 SDS 页分析样本。</li>
		<li>进行样品的化验或贮存。</li>
	</ol>
</li>
</ol>

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作者没有披露。

虽然添加 polyhistidine 标签的重组蛋白和其纯化的 imac 已经成为几乎无处不在的生物化学文献3,4,5, IMAC 在酶纯化中的应用在生物化学教学实验室中仍然稀少, 并且出版的方法不总考虑教学实验室的资源限制20。此外, 在教学实验室中使用 imac 是最有效的, 当结合实验, 评估活动和纯度, 使 imac 纯化酶的一种理想的教学活动。为了将 IMAC 的应用推广到酶的纯化, 包括 metalloenzymes, 在教学实验室中, 需要可靠、廉价的方法。在本协议中, 我们展示了台式 imac 使用现成的和廉价的实验室用品, 同时也解决了在应用 IMAC 到金属蛋白22的限制, 通过重组铁 (II) 依赖金属酶, 左旋多巴酶, 后净化。使用所描述的试剂和材料, 我们估计八个学生群体的消耗品成本是每学期500-600 元, 以运行此协议, 包括图 1和图 2中概述的分析步骤。
由于 fe2 +可与氨基酸配体24分离, 铁2 + 2至 fe3 +的简便氧化, 重组金属酶非血红素、氨基酸螯合铁 (II.)酶纯化的典型成分。当使用经典色谱法时, 在某些情况下可以避免铁的总损失32, 但更常见的是, 铁 (II) 在还原剂33、34、35的存在下被加回,36经常在厌氧气氛37,38,39, 并且在某些情况下多余的铁不被去除33,34,36, 复杂化任何后续化验。经典色谱和厌氧气氛的连续步骤对于本科实验室来说并不现实, 促使了该协议的发展。
虽然 NTA 柱的手工制备和大量使用重力的样品的处理确实需要额外的时间和精力, 但与预包装的柱和自动色谱仪相比, 手动步骤允许动手由学生学习, 从而加深对过程背后的科学的理解。在这里概述的条件下添加铁 (II) 盐对过量的 dithiothreitol 特别敏感。如果学生误加过量的 dithiothreitol, 可能会发生降水事件。我们发现, 要求学生在到达实验室之前对试剂数量进行计算是有帮助的, 所以实验室时间可以在板凳上最有效地使用。整个台式 IMAC 纯化-从细胞裂解到蛋白洗脱-可以完成在一个4小时的实验室期间, 然后重建和化验在随后的实验室期间。

第一个报告从寄主有机体提取物中纯化 polyhistidine 标记蛋白, 使用固定化金属螯合的组氨酸标记进入文献在 1988年1,2。从那时起, 在生化文献3、4中, 对重组蛋白添加 polyhistidine 标记, 并通过固定化的金属亲和层析 (IMAC) 进行纯化已成为几乎无处不在的. 5。IMAC 纯化方法可以在台式上实现, 使用自动色谱和自旋柱格式。亲和纯化方法, 特别是 IMAC, 在研究实验室中得到广泛应用, 在本科教学实验室中的应用较少。最广泛使用的实验室教科书为生物化学实验室不常规地教这些方法, 相反选择更加传统的离子交换或染料结合色谱6,7,8,9. 例如, 安德森10对乳酸脱氢酶的纯化使用了染料结合的亲和性, 而牛奶α-乳白蛋白7、11的纯化博耶使用镍 nitriloacetic酸基质, 但没有重组的聚组氨酸标签, 依靠而不是内在亲和力的蛋白质为树脂。一些现代本科生实验室教科书和出版物对聚组氨酸标记蛋白靶 (如绿色或红色荧光蛋白12,13) 实施固定化金属亲和层析, 14、15、抗体16, 并选择酶17、18、19、20, 甚至一些未知功能21。可以说, 在教学实验室中, 一种酶的纯化是可取的, 因为在以后的课程中, 目标能够被检测出来, 丰富了学生的 "真正科学" 的经验;事实上, 这些类型的实验室经验已经发表, 学生学习的有益结果报告了17,18,20,21。然而, 在生物化学教学实验室中, IMAC 在酶纯化中的应用仍然稀少, 而且公布的方法甚至可以推测出在课堂实验室中通常无法使用的色谱仪器。20. IMAC 在金属蛋白中的应用也有局限性, 特别是那些束缚氧化还原敏感的价金属对活动22至关重要的因素。通常, 金属离子在纯化过程中丢失或氧化, 产生一种不适合于本科生实验室的非活性酶。
一整1/3 的酶结合金属离子23, 尽管几乎普遍要求铁在所有形式的生命23, 铁可以说是最有问题的金属离子在酶学中。非血红素 Fe2 +结合酶特别容易在 IMAC 中的金属损耗和/或氧化;大概是由于缺乏一个专门的有机配体如血红素和容易与 Fe2 +可以离解从氨基酸配位24。此外, 由于负自由能的变化和铁3 +的相对稳定性, fe2 +对 fe3 +的氧依赖性氧化是自发的水溶液。通常, 这些挑战是通过使用厌氧大气和/或非 IMAC 色谱方法22来克服的。在本文中, 我们将演示使用台式 IMAC 净化 Fe2 +依赖金属酶 l-多巴酶使用简单, 廉价的色谱耗材, 然后重组的活动现场 Fe2 +, 并酶法测定。这些方法在我们自己的本科生生物化学实验室6-12 个学生小组是标准的, 并且可以被使用扩展酶调查的节目在本科生水平上。

<table><tbody><tr><td>consumables</td><td></td><td></td><td></td></tr><tr><td>BeadBeater 0.1mm glass beads</td><td>BioSpec Products</td><td>11079101</td><td>1 pound each</td></tr><tr><td>50mL Conical Tubes with Screw Caps, sterile</td><td>VWR</td><td>21008-178</td><td></td></tr><tr><td>Sodium chloride</td><td>Fisher Bioreagents</td><td>BP358-1</td><td></td></tr><tr><td>Potassium phosphate, monobasic</td><td>Acros (Fisher)</td><td>AC42420-5000</td><td></td></tr><tr><td>Sodium Ascorbate</td><td>Acros (Fisher)</td><td>AC35268-1000&amp;nbsp;</td><td></td></tr><tr><td>DTT (Dithiothreitol)</td><td>Lab Scientific</td><td>D-115</td><td></td></tr><tr><td>Iron(II) sulfate heptahydrate</td><td>Sigmaaldrich</td><td>310077</td><td></td></tr><tr><td>HisPur NiNTA Resin&amp;nbsp;</td><td>Fisher (pierce)</td><td>PI88221</td><td></td></tr><tr><td>Econo-Column Chromatography Columns - 1.5 x 20cm</td><td>Bio-Rad</td><td>737-1522&amp;nbsp;</td><td>1.5 x 20 cm, 35 ml, 2ea,&amp;nbsp;</td></tr><tr><td>Stopcock Valve, one way, female to male luer</td><td>Kimble</td><td>420163-0000</td><td>pack of 50</td></tr><tr><td>BD 10mL luer-loc syringe (non-sterile, without needle)</td><td>VWR</td><td>301029</td><td></td></tr><tr><td>Fitting for tubing: 1.6 mm Barb to Female Luer</td><td>Biorad</td><td>7318222</td><td></td></tr><tr><td>Fitting for tubing: 1.6 mm Barb to Male Luer&amp;nbsp;</td><td>Biorad</td><td>7318225</td><td></td></tr><tr><td>Silicon Tubing (1.6 mm ID/0.8 mm wall, for 0.2-5 ml/min on Peristaltic Pump)</td><td>Bio-Rad</td><td>7318211</td><td>Pkg of 1, 1.6 mm ID/0.8 mm wall, 10 m, low-pressure tubing for liquid handling</td></tr><tr><td>Glycerol</td><td>Fisher (Pierce)</td><td>17904</td><td></td></tr><tr><td>Coomassie Plus (Bradford) Protein Assay</td><td>Thermo Scientitic</td><td>23236</td><td></td></tr><tr><td>Microcentrifuge Tubes, snap cap, 1.5mL</td><td>VWR</td><td>89000-028</td><td></td></tr><tr><td>Fisherbrand polystyrene disposable serological pipets</td><td>Fisher</td><td>13-676-10F</td><td></td></tr><tr><td>Fiserbrand universal pipet pump</td><td>Fisher&amp;nbsp;</td><td>14-955-110</td><td></td></tr><tr><td>Fisherbrand Transfer Pipets</td><td>Fisher&amp;nbsp;</td><td>13-711-9AM</td><td></td></tr><tr><td>Econo-Pac 10DG desalting columns</td><td>Bio-Rad</td><td>732-2010</td><td>box of 30</td></tr><tr><td>ExpressPlus PAGE 5x sample buffer</td><td>Genscript</td><td>MB01015</td><td>5mL (Dilute 1:5 with sample)</td></tr><tr><td>ExpressPlus PAGE Gel, 4-20%, 12 wells</td><td>Genscript</td><td>M42012</td><td>20 gels</td></tr><tr><td>Fisherbrand Disposable Cuvettes, Methyacrylate</td><td>Fisher</td><td>14-955-128&amp;nbsp;</td><td>case of 500</td></tr><tr><td>Cuvette Caps Square Disposable</td><td>Fisher</td><td>14-385-999</td><td></td></tr><tr><td>L-DOPA (3,4-dihydroxyphenylalanine)</td><td>Acros</td><td>D9628-5G</td><td></td></tr><tr><td>Permanent Equipment:&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>BeadBeater 50mL chamber&amp;nbsp;</td><td>BioSpec Products</td><td>110803-50SS</td><td>1 chamber</td></tr><tr><td>BeadBeater</td><td>BioSpec Products</td><td>1107900-101</td><td>350 ml polycarbonate chamber, rotor assembly, motor base, ice-water cooling jacket and one pound of glass beads.</td></tr><tr><td>Centrifuge tubes, High-Speed PPCO, 50mL</td><td>Fisher</td><td>3119-0050PK</td><td></td></tr><tr><td>Mini-PROTEAN Tetra Vertical Electrophoresis Cell for Mini Precast Gels, 4-gel</td><td>Bio-Rad</td><td>1658004</td><td>&amp;nbsp;4-gel vertical electrophoresis system, includes electrode assembly, companion running module, tank, lid with power cables, mini cell buffer dam</td></tr><tr><td>PowerPac HC Power Supply</td><td>Bio-Rad</td><td>1645052</td><td>100&amp;ndash;120/220&amp;ndash;240 V, power supply for high-current applications, includes power cord</td></tr><tr><td>UV-1800 with UV-Probe Software</td><td>Shimadzu</td><td>UV-1800</td><td></td></tr><tr><td>Kintek Global Kinetic Explorer</td><td>Kintek Corp</td><td>version 6</td><td>https://www.kintekexplorer.com/downloads/</td></tr></tbody></table>

benchtop immobilized metal affinity chromatography, reconstitution and assay of a polyhistidine tagged metalloenzyme for the undergraduate laboratory

台式固定化金属亲和层析 (IMAC), polyhistidine 标记蛋白易于掌握的本科生和已成为最广泛使用的蛋白质纯化方法在现代文学。但是, 亲和层析技术应用于金属结合蛋白, 特别是那些与氧化还原敏感金属 (如铁) 有关的实验室, 通常只限于进入手套箱的化验室--在本科生中通常不具备这种设备。实验室。在本文中, 我们展示了我们的台式方法的分离, IMAC 纯化和金属离子重组的多组氨酸标记, 氧化还原活性, 非血红素铁结合 extradiol 酶和分析酶的不同基质浓度和饱和氧。这些方法由本科生执行, 并在本科教学和研究实验室实施, 并在主要本科院校提供方便和负担得起的仪器。

这些代表性的结果是由本科生收集, 因为他们执行本议定书在两个课程实验室期间的 341: 实验生物化学在 Muhlenberg 学院。图 1展示了 20 kDa 聚组氨酸标记金属酶、l-多巴酶的纯化结果, 这是由两名本科生在4小时的课堂实验室期间进行的, 并通过 SDS 页进行分析。学生在随后的实验室期间。对聚组氨酸标记蛋白进行有效纯化 (图 1, 车道 2)。用抗体对多组氨酸标记的应用免疫印迹表明, 少量的多组氨酸标记靶蛋白在流动中丢失 (未显示数据), 可能是因为在中的目标蛋白超过100毫克裂解液超过了树脂的粘结能力。
图 2展示了学生收集的活动数据, 这些酶反应的聚组氨酸标记金属酶靶, l-多巴酶, 重组后与铁 (II) 和随后的凝胶过滤, 如协议所述此处.在收集数据之前的五秒死亡时间是第一次执行此项技术的学生的典型。用已发表的试验检测了金属酶的鲁棒活性, 并产生了与公布结果一致的稳态动力学参数29。通过拟合图 2所示的进度曲线30 , 确定了稳态动力学参数;然而, 在一系列基底浓度下收集的初始速率的非线性最小二乘拟合同样可能是31。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58012/58012fig1.jpg"> 图1。SDS-页26对聚组氨酸标记蛋白在纯化前、中、后的分析。车道 1-分子量标记, 2 纯化蛋白 (20 kDa) 后镍 NTA, 3 镍 NTA 流经, 4-细胞游离粗萃取物纯化前, 5 细胞碎片颗粒后裂解。采用5x 试样/加载缓冲剂, 用凝胶电泳系统对预铸4-20% 聚丙烯酰胺凝胶进行分离。<a href="https://www.jove.com/files/ftp_upload/58012/58012fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58012/58012fig2.jpg"> 图2。金属酶重组铁 (II.) 的稳态测定 (即, l-多巴酶, 10µM) 与基板 (l-多巴-5 µM (红色), 25 µM (绿色), 50 µM (蓝色)) 在缓冲 (50 毫米磷酸盐, 200 毫米氯化钠, pH 8)。痕迹描绘的产品形成在414nm。利用1毫升甲基丙烯酸小试管在分束扫描紫外可见光谱仪29中连续获得吸光度数据。原始数据适合模型的米氏-米氏稳态逼近 (kM 30.8 µM 14.4, k猫明显2.3 s-1 @ 0.05)30。<a href="https://www.jove.com/files/ftp_upload/58012/58012fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Benchtop Immobilized Metal Affinity Chromatography, Reconstitution and Assay of a Polyhistidine Tagged Metalloenzyme for the Undergraduate Laboratory at JoVE.

Benchtop Immobilized Metal Affinity Chromatography, Reconstitution and Assay of a Polyhistidine Tagged Metalloenzyme for the Undergraduate Laboratory

在这里, 我们提出了一个协议, 台式固定化金属亲和层析纯化和随后重组的 polyhistidine 标签, 非血红素铁结合酶适合本科教学实验室。

台式固定化金属亲和层析、Polyhistidine 标记金属酶的重组与检测

Benchtop immobilized metal affinity chromatography (IMAC), of polyhistidine tagged proteins is easily mastered by undergraduate students and has become ...

benchtop-immobilized-metal-affinity-chromatography-reconstitution

Research

JoVE Journal

Biochemistry

17.2K 次观看.  Muhlenberg College. 在这里, 我们提出了一个协议, 台式固定化金属亲和层析纯化和随后重组的 polyhistidine 标签, 非血红素铁结合酶适合本科教学实验室。

视频: Benchtop Immobilized Metal Affinity Chromatography, Reconstitution and Assay of a Polyhistidine Tagged Metalloenzyme for the Undergraduate Laboratory

Iridium(III) Luminescent Probe for Detection of the Malarial Protein Biomarker Histidine Rich Protein-II

This work outlines the synthesis of a non-emissive, cyclometalated Ir(III) complex, Ir(ppy)2(H2O)2+ (Ir1), which elicits a rapid, long-lived phosphorescent signal when coordinated to a histidine-containing protein immobilized on the surface of a magnetic particle. Synthesis of Ir1, in high yields,is complete O/N&#160;and involves splitting of the parent cyclometalated Ir(III) chloro-bridged dimer into two equivalents of the solvated complex. To confirm specificity, several amino acids were probed for coordination activity when added to the synthesized probe, and only histidine elicited a signal response. Using BNT-II, a branched peptide mimic of the malarial biomarker Histidine Rich Protein II (pfHRP-II), the iridium probe was validated as a tool for HRP-II detection. Quenching effects were noted in the BNT-II/Ir1 titration when compared to L-Histidine/Ir1, but these were attributed to steric hindrance and triplet state quenching. Biolayer interferometry was used to determine real-time kinetics of interaction of Ir1 with BNT-II. Once the system was optimized, the limit of detection of rcHRP-II using the probe was found to be 12.8 nM in solution. When this protein was immobilized on the surface of a 50 &#181;m magnetic agarose particle, the limit of detection was 14.5 nM. The robust signal response of this inorganic probe, as well as its flexibility of use in solution or immobilized on a surface, can lend itself toward a variety of applications, from diagnostic use to imaging.

IridiumIII Luminescent Probe for Detection of the Malarial Protein Biomarker Histidine Rich Protein-II

Robust detection reagents are of increasing necessity for developing new malaria diagnostic tools. An iridium(III) probe was designed that emits long-lasting luminescent signal in the presence of a histidine-rich malarial protein biomarker. Detection of the protein either in solution or immobilized on a magnetic particle affords flexibility in application.

铱（III）荧光探针检测疟疾蛋白质生物标志物组氨酸丰富的蛋白质-Ⅱ

This work outlines the synthesis of a non-emissive, cyclometalated Ir(III) complex, Ir(ppy)2(H2O)2+ (Ir1), which elicits a rapid, long-lived ...

科研

化学

Method for Efficient Refolding and Purification of Chemoreceptor Ligand Binding Domain

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be greatly facilitated by the production of milligram amounts of pure, folded ligand binding domains. Attempts to heterologously express periplasmic ligand binding domains of bacterial chemoreceptors in Escherichia coli (E. coli) often result in their targeting into inclusion bodies. Here, a method is presented for protein recovery from inclusion bodies, its refolding and purification, using the periplasmic dCACHE ligand binding domain of Campylobacter jejuni (C. jejuni) chemoreceptor Tlp3 as an example. The approach involves expression of the protein of interest with a cleavable His6-tag, isolation and urea-mediated solubilisation of inclusion bodies, protein refolding by urea depletion, and purification by means of affinity chromatography, followed by tag removal and size-exclusion chromatography. The circular dichroism spectroscopy is used to confirm the folded state of the pure protein. It has been demonstrated that this protocol is generally useful for production of milligram amounts of dCACHE periplasmic ligand binding domains of other bacterial chemoreceptors in a soluble and crystallisable form.

A procedure is presented for the refolding of the dCACHE periplasmic ligand binding domain of Campylobacter jejuni chemoreceptor Tlp3 from inclusion bodies and the purification to yield milligram quantities of protein.

化学感受器配体结合域的高效复用与纯化方法

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be ...

生物化学

Ion Mobility-Mass Spectrometry Techniques for Determining the Structure and Mechanisms of Metal Ion Recognition and Redox Activity of Metal Binding Oligopeptides

Electrospray ionization (ESI) can transfer an aqueous-phase peptide or peptide complex to the gas-phase while conserving its mass, overall charge, metal-binding interactions, and conformational shape. Coupling ESI with ion mobility-mass spectrometry (IM-MS) provides an instrumental technique that allows for simultaneous measurement of a peptide&#8217;s mass-to-charge (m/z) and collision cross section (CCS) that relate to its stoichiometry, protonation state, and conformational shape. The overall charge of a peptide complex is controlled by the protonation of 1) the peptide&#8217;s acidic and basic sites and 2) the oxidation state of the metal ion(s). Therefore, the overall charge state of a complex is a function of the pH of the solution that affects the peptides metal ion binding affinity. For ESI-IM-MS analyses, peptide and metal ions solutions are prepared from aqueous-only solutions, with the pH adjusted with dilute aqueous acetic acid or ammonium hydroxide. This allows for pH dependence and metal ion selectivity to be determined for a specific peptide. Furthermore, the m/z and CCS of a peptide complex can be used with B3LYP/LanL2DZ molecular modeling to discern binding sites of the metal ion coordination and tertiary structure of the complex. The results show how ESI-IM-MS can characterize the selective chelating performance of a set of alternative methanobactin peptides and compare them to the copper-binding peptide methanobactin.

Ion mobility-mass spectrometry and molecular modeling techniques can characterize the selective metal chelating performance of designed metal-binding peptides and the copper-binding peptide methanobactin. Developing new classes of metal chelating peptides will help lead to therapeutics for diseases associated with metal ion misbalance.

用于测定金属结合寡聚肽的金属电子识别和机制的Ion流动性-质谱技术

Electrospray ionization (ESI) can transfer an aqueous-phase peptide or peptide complex to the gas-phase while conserving its mass, overall charge, ...

Quantification of Metal Leaching in Immobilized Metal Affinity Chromatography

Contamination of enzymes with metals leached from immobilized metal affinity chromatography (IMAC) columns poses a major concern for enzymologists, as many of the common di-and trivalent cations used in IMAC resins have an inhibitory effect on enzymes. However, the extent of metal leaching and the impact of various eluting and reducing reagents are poorly understood in large part due to the absence of simple and practical transition metal quantification protocols that use equipment typically available in biochemistry labs. To address this problem, we have developed a protocol to quickly quantify the amount of metal contamination in samples prepared using IMAC as a purification step. The method uses hydroxynaphthol blue (HNB) as a colorimetric indicator for metal cation content in a sample solution and UV-Vis spectroscopy as a means to quantify the amount of metal present, into the nanomolar range, based on the change in the HNB spectrum at 647 nm. While metal content in a solution has historically been determined using atomic absorption spectroscopy or inductively coupled plasma techniques, these methods require specialized equipment and training outside the scope of a typical biochemistry laboratory. The method proposed here provides a simple and fast way for biochemists to determine the metal content of samples using existing equipment and knowledge without sacrificing accuracy.

We present an assay for easy quantification of metals introduced to samples prepared using immobilized metal affinity chromatography. The method uses hydroxynaphthol blue as the colorimetric metal indicator and a UV-Vis spectrophotometer as the detector.

金属浸出在固定金属亲和力色谱中的定量

Contamination of enzymes with metals leached from immobilized metal affinity chromatography (IMAC) columns poses a major concern for enzymologists, as ...

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude stringent methods for the isolation and identification of protein networks around a protein of interest. The use of chemical crosslinkers allows the selective stabilization of labile interactions, thus bypassing biochemical limitations for purification. Here we present a protocol amenable for cells in culture that uses a homobifunctional crosslinker with a spacer arm of 12 &Aring;, dithiobis-(succinimidyl proprionate) (DSP). DSP is cleaved by reduction of a disulphide bond present in the molecule. Cross-linking combined with immunoaffinity chromatography of proteins of interest with magnetic beads allows the isolation of protein complexes that otherwise would not withstand purification. This protocol is compatible with regular western blot techniques and it can be scaled up for protein identification by mass spectrometry1.
Stephanie A. Zlatic and Pearl V. Ryder contributed equally to this work.

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The cell permeable crosslinker DSP [dithiobis-(succinimidyl propionate)] stabilizes transient and labile interactions in vivo, which allows their isolation using stringent protein complex purification techniques. Here we present a technique for crosslinking cells grown in culture followed by isolation of protein complexes by immunoprecipitation.

由不稳定的多蛋白质复合物的分离在体内控制细胞的交联和免疫磁性亲和层析

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude ...

生物学

使用生物物理测定表征 PROTAC 中三元复合物的形成

The Development and Application of Biophysical Assays for Evaluating Ternary Complex Formation Induced by Proteolysis Targeting Chimeras (PROTACS)

E3 ligases and proteins targeted for degradation can be induced to form complexes by heterobifunctional molecules in a multi-step process. The kinetics and thermodynamics of the interactions involved contribute to efficiency of ubiquitination and resulting degradation of the protein. Biophysical techniques such as surface plasmon resonance (SPR), biolayer interferometry (BLI), and isothermal titration calorimetry (ITC) provide valuable information that can be used in the optimization of those interactions. Using two model systems, a biophysical assay tool kit for understanding the cooperativity of ternary complex formation and the impact of the &#39;hook effect&#39; on binding kinetics was established. In one case, a proteolysis targeting chimera (PROTAC) molecule that induced ternary complex formation between Brd4BD2 and VHL was evaluated. The heterobifunctional molecule, MZ1, has nM affinities for both the Brd4BD2 protein (SPR KD = 1 nM, ITC KD = 4 nM) and the VHL complex (SPR KD = 29 nM, ITC KD = 66 nM). For this system, robust SPR, BLI, and ITC assays were developed that reproduced published results demonstrating the cooperativity of ternary complex formation. In the other case, a molecule that induced ternary complexes between a 46.0 kDa protein, PPM1D, and cereblon [CRBN (319-442)] was studied. The heterobifunctional molecule, BRD-5110, has an SPR KD = 1 nM for PPM1D but much weaker binding against the truncated CRBN (319-442) complex (SPR KD= ~ 3 &#181;M). In that case, the binding for CRBN in SPR was not saturable, resulting in a &#34;hook-effect&#34;. Throughput and reagent requirements for SPR, BLI, and ITC were evaluated, and general recommendations for their application to PROTAC projects were provided.

作者聚焦：评估用于表征 PROTACS 三元复合物的生物物理检测 

Here we describe protocols for the biophysical characterization of ternary complex formation induced by proteolysis targeting chimeras (PROTACS) that involve the ubiquitin ligases Von Hippel-Lindau E3 ligase (VHL) and Cereblon (CRBN). Biophysical methods illustrated herein include surface plasmon resonance (SPR), biolayer interferometry (BLI), and isothermal titration calorimetry (ITC).

用于评估蛋白水解靶向嵌合体（PROTACS）诱导的三元复合物形成的生物物理测定方法的发展和应用

E3 ligases and proteins targeted for degradation can be induced to form complexes by heterobifunctional molecules in a multi-step process. The kinetics ...

<meta charset="utf8"></head><body>优化 FPLC 以高效纯化 His 标记的 FEN1 蛋白

Affinity Purification of a 6X-His-Tagged Protein using a Fast Protein Liquid Chromatography System

Functional characterization of proteins requires them to be expressed and purified in substantial amounts with high purity to perform biochemical assays. The Fast Protein Liquid Chromatography (FPLC) system allows high-resolution separation of complex protein mixtures. By adjusting various parameters in FPLC, such as selecting the appropriate purification matrix, regulating the protein sample's temperature, and managing the sample's flow rate onto the matrix and the elution rate, it is possible to ensure the protein's stability and functionality. In this protocol, we will demonstrate the versatility of the FPLC system to purify 6X-His-tagged flap endonuclease 1 (FEN1) protein, produced in bacterial cultures. To improve protein purification efficiency, we will focus on multiple considerations, including proper column packing and preparation, sample injection using a sample loop, flow rate of sample application to the column, and sample elution parameters. Finally, the chromatogram will be analyzed to identify fractions containing high yields of protein and considerations for proper recombinant protein long-term storage. Optimizing protein purification methods is crucial for improving the precision and reliability of protein analysis.

<meta charset="utf8"></head><body>作者聚焦:优化 His 标记的 FEN1 蛋白的亲和层析

This article provides a procedure for the affinity purification of a human recombinant protein, flap endonuclease 1 (FEN1), which has been labeled with a 6X-histidine tag. The protocol involves the utilization of two distinct immobilized metal ion columns for the purification of the tagged protein.

使用 Fast Protein 液相色谱系统对 6X-His 标签蛋白进行亲和纯化 

Functional characterization of proteins requires them to be expressed and purified in substantial amounts with high purity to perform biochemical assays. ...

台式固定化金属亲和层析、Polyhistidine 标记金属酶的重组与检测

摘要

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此视频中的章节

铱（III）荧光探针检测疟疾蛋白质生物标志物组氨酸丰富的蛋白质-Ⅱ

化学感受器配体结合域的高效复用与纯化方法

用于测定金属结合寡聚肽的金属电子识别和机制的Ion流动性-质谱技术

金属浸出在固定金属亲和力色谱中的定量

由不稳定的多蛋白质复合物的分离在体内控制细胞的交联和免疫磁性亲和层析

用于评估蛋白水解靶向嵌合体（PROTACS）诱导的三元复合物形成的生物物理测定方法的发展和应用

用于评估蛋白水解靶向嵌合体（PROTACS）诱导的三元复合物形成的生物物理测定方法的发展和应用

用于评估蛋白水解靶向嵌合体（PROTACS）诱导的三元复合物形成的生物物理测定方法的发展和应用

使用 Fast Protein 液相色谱系统对 6X-His 标签蛋白进行亲和纯化

使用 Fast Protein 液相色谱系统对 6X-His 标签蛋白进行亲和纯化