Name: biochemical and structural characterization of the carbohydrate transport substrate-binding-protein sp0092
Uploaded: 2017-10-02T15:00:00.000+00:00
Duration: 8 min 53 s
Description: Watch this Scientific Journal Video about Biochemical and Structural Characterization of the Carbohydrate Transport Substrate-binding-protein SP0092 at JoVE.com

我们承认 OPPF-英国在克隆技术方面的帮助, 哈里斯为 SEC 购物中心和光束 I03 和 I04 在钻石光源的科学家。

注意: 信号肽被删除的编码序列在标准的融合协议后的 pOPINF 向量中被克隆; 本机蛋白在大肠杆菌中被表达为他的标记融合 大肠埃希氏大肠杆菌 BL21 罗塞塔细胞 28 , 29 . 硒标记的变体按照制造商 30 的标准方法表示。重组的收缩蛋白被纯化为先前描述的 31 , 32 。
 1. 生化特性 <ol> <li> 热转移检测 <ol> <li> 准备48个具有 pH 范围的缓冲液, 从4.0 到 9.5, 氯化钠浓度从0到0.5 米, 并分配40和 #181; 在96井板中描述 表 1 33 . </li> <li> 添加到每个井5和 #181; 在 1-2 毫克/毫升浓度的情况下, 该方案的 L. </li>
		<li> 添加到每个井5和 #181; L 20x 和 #160; 蛋白质的荧光染色 (请参阅 材料表 ). </li>
		<li> 将解决方案由移混合, 用透明的粘合剂膜密封该板, 室温下在 112 x g 处旋转2分钟. </li>
		<li> 在 real-time 机上运行实验: 设置3和 #176 的温度斜坡; c/分钟从4到99和 #176; c 与十年代保持时间, 并且读荧光每0.5 和 #176; c 与励磁在483毫微米和放射在568毫微米. </li>
		<li> 分析每个缓冲条件的荧光发射曲线, 确定熔化温度 (T m ) 作为导数的最小值. 
		注意: 这个过程提供了最好的缓冲液, 以帮助改善蛋白质的稳定性。根据具有最高 T m 的缓冲条件选择 pH 值范围和盐浓度, 并添加 2.5% (v/v) 的甘油和0.5 毫米的三 (2-乙) 膦 (TCEP), 以定义以下步骤的缓冲液 (秒缓冲). </li>
	</ol> </li> <li> 加工和分析秒 <ol> <li> 使用脱过滤水的包装凝胶过滤柱进行两柱体洗涤 (用宽范围的分子量 24 mL 柱). </li>
		<li> 将柱形连接到光散射检测器上, 并平衡从热位移检测中确定的秒缓冲柱. </li>
		<li> 注入100和 #181; 在5毫克/毫升的纯化的平衡秒列, 并流一列的 sec 缓冲区的体积. </li>
		<li> 检查洗脱色谱是否为单个或多个峰值, 并使用分析软件检测散射数据, 获取所有物种的摩尔质量和多指数. 
		注: 对于每一个聚物种的峰值, 在质量/摩尔质量加权的摩尔质量 (兆瓦/锰) 上, 对多指数进行检验是有益的。接近1的多值表示分散峰值. 
		注意: 一旦所有的齐聚物种被定义, 研究它们对蛋白质浓度的依赖性是有用的, 因为较高的浓度可能有利于更大的齐聚物形成. </li>
		<li> 执行多个分析秒运行; 注入100和 #181; 向平衡凝胶过滤塔 (0.1-10 毫克/毫升) 增加浓度 (使用宽范围的分子量 5 ml 柱), 每一次流一列的秒缓冲液. </li>
		<li> 检查洗脱色谱的单个或多个峰值, 并分析与不同起始蛋白质浓度对应的 280 nm 曲线的吸光度强度。如果不同峰的相对强度保持不变, 则不存在聚物种之间的改制。不同浓度的相对强度的变化表明, 不同聚物种之间的转换依赖于蛋白质浓度. 
		注意: 这个表征步骤是定义哪些物种更适合于结晶的基础。事实上, 不同的分散物种更容易结晶, 如果它们是在一个稳定的聚状态在一个明确的浓度. </li>
	</ol> </li> </ol> 2。蛋白质制备与结晶 <ol> <li> 准备秒 <ol> <li> 在用脱过滤水进行一柱式容积洗涤后, 平衡用秒缓冲剂制备的包装凝胶过滤柱 (宽范围分子量120毫升列). </li>
		<li> 向平衡凝胶过滤柱注入 1-5 毫升的纯化的收缩剂, 并将一柱流量的秒数缓冲. </li>
		<li> 以1毫升的分数收集列流. </li>
		<li> 将与单个单分散峰值相对应的分数汇集在一起; 在15毫升离心过滤器中旋转1500克的样品, 并将浓度量化, 直到达到所需浓度 (通常为 SP0092 的 50-100 毫克/毫升). </li>
	</ol> </li> <li> 结晶 <ol> <li> 使用制造商描述的预结晶试验确定结晶试验的最佳浓度范围: <ol> <li> 0.5-1.0 毫升每四结晶试验解决方案在一个不同的油藏24井下落结晶板. </li>
			<li> 将1和 #181 的蛋白质溶液与1和 #181 的溶出液混合, 在室温下孵育不少于1小时 (在夜间孵化后获得更可靠的结果). </li>
			<li> 使用10x 和 #160 检查每个浓度的跌落质量; 放大显微镜 
			注: 测试至少三种不同的蛋白质浓度: (I) 大部分残留的水滴清楚表明, 蛋白质太稀, 无法结晶;(二) 在大多数下落中出现大量沉淀, 意味着浓度过高;和 (III) 平衡的发生, 无论是明确的下降和沉淀 (更好的, 如果光) 是一个很好的迹象表明, 测试浓度有利于结晶. </li>
		</ol> </li> <li> 准备96个井下的下落结晶板; 分配100和 #181; 在每个油藏中不同的商业结晶溶液 L 34 , 35 , 36 , 37 . </li> <li> 使用晶化机器人系统在所有的结晶滴井中, 在先前定义的浓度 (步骤 1.1.6) 中分配 100 nL 蛋白质样本. </li>
		<li> 将 100 nL 不同的油藏溶液分配给相应的结晶滴井, 与步骤2.2.3 中的蛋白质混合。密封的结晶板, 以避免蒸发, 使平衡的结晶下降与水库. </li>
		<li> 每 1-2 天, 每个星期晚些时候) 使用10x 和 #160;(至少) 放大显微镜来评估晶体的形成和生长. 
		注: 用于蛋白质结晶的自动成像系统可用于水滴可视化和捕获. </li>
	</ol> </li> </ol> p 类 = "jove_title ">> 3。晶体特性和 x 射线数据收集 <ol> <li> 晶体安装 <ol> <li> 准备剂溶液, 方法是将甘油 (最终浓度) 的 25% (v/v) 添加到结晶条件 (从而在初始状态下替换25% 的水混合) 38 . </li>
		<li> 用液氮填充泡沫杜瓦, 并将单冰球的样品箱部分放入杜瓦瓶中。让它冷却到液氮温度. </li>
		<li> 切割和从结晶板上取下密封胶带, 在晶体形成的下落处. </li>
		<li> 将一滴1和 #181; 剂解决方案放置在靠近目标下落的 coverslide 上. </li>
		<li> 将所选的晶体从原始下落转移到剂溶液下落使用在磁性棒上安装的脊柱标准底座上的尼龙冷冻环 39 , 40 . </li>
		<li> 快速将晶体从剂滴转移到液氮, 将该回路放入单冰球样品架的第一个空位置. </li>
		<li> 重复 (从密封胶带切割步骤), 直到所有所需的晶体收获和存储在单冰球样品持有人. </li>
		<li> 使用冰球棒将单冰球基地放在单冰球上, 然后将冰球带到光束 (在液氮条件下). 
		注意: 温度极低! </li>
		<li> 使用冷冻钳和冰球杜瓦加载工具加载的单冰球 (s) 到光束样品更换机器人杜瓦瓶。单冰球样品持有人将分离从基地盖子离开样本循环持有人直立在样品机器人杜瓦, 从而暴露在液氮和可访问的样品更换机器人. </li>
	</ol> </li> <li> 晶体特性 注意: 可以使用实验室 x 射线源或同步辐射 (SR) x 射线源对收获的晶体进行衍射质量筛选。高分子晶体学 (MX) 光束提供了一种可调谐的能量源来利用反常衍射进行结构解。在下面的部分中, 提出了一系列的工作指示, 与实验能力的钻石光源 mx 光束, 但这些准则也可以适应 mx 光束在其他加速器世界各地。
	<ol> <li> 作为第一步, 确认或识别晶体中的反常散射是很有用的。通过测量晶体的 x 射线荧光光谱可以方便地实现这一目标。首先确保光束的 X 射线能量设置得足够高, 以激发大分子晶体 (14 凯文或更高) 通常预期的所有元素. </li>
		<li> 使用光束控制软件选择由采样转换器安装的样品; 回路和 #160; 将自动集中在 X 射线束上, 并可手动确认晶体定心, #160; 使用光束控制软件. </li>
		<li> 使用光束控制软件记录 X 射线荧光光谱: 用户选择曝光时间并启动测量 (荧光/荧光光谱设置, 和 #8594; 运行, 图 2A )。光束控制软件自动定位 x 射线荧光探测器到位, 并确定最小入射 x 射线通量, 以获得在荧光探测器上的可读信号。然后以自动的方式分析获得的频谱, 并将发射峰安装在自然发生的生物元素上。这些例程可在我们公司的光束控制软件图形用户界面 (GUI)-GDA (www.opengda.org) 中获得, 在整个欧洲加速器 41 , 42 . </li>
		<li> 如果确定了可用于反常衍射实验的合适元素, 则执行 X 射线吸收边缘能量扫描, 以确定从晶体中收集异常衍射数据的最佳波长: 在光束上控制软件, 选择元素并启动扫描 (荧光/荧光扫描设置, 和 #8594; Atom 名称, 和 #8594; 运行, 图 2B ). 
		注: 对于一个单一的反常衍射实验, 吸收边缘的峰值被用来最大化的反常散射。进行多波长反常衍射实验的实验考虑以前是详细的 43 . </li>
		<li> 要确定晶体单元的单元参数、对称性和衍射极限, 请在45和 #176 测量三 x 射线衍射图样; 使用振荡方法的间隔 (数据收集/筛选和 #160; #8594; 运行当前, 图 2C )。光束控制软件为用户提供了一个振荡角度和曝光时间的选择, 并能够设定 X 射线束的透射率。对于标准的晶体筛查, 建议了0.5 和 #176 的振荡角; 0.5 秒的曝光时间和 5% X 射线束传输。所测得的衍射图像由埃德娜管道自动分析, 并返回一组完整的数据集 22 的集合策略. </li>
	</ol> </li> <li> X 射线数据收集 注意: 用户可以选择在标准振荡模式或逆波束模式下收集数据, 这使弗里德尔的队友能够在时间和 X 射线剂量之间进行记录, 并具有近似相同的吸收行为允许更精确地测量反常的差异。后者是有用的, 特别是如果预期小的反常差异和/或样品是辐射敏感的, 当进行一个反常的衍射实验。有关使用的最佳数据收集策略的注意事项已被全面审阅 22 , 23 , 44 , 45 , 46 , 47 . <ol> <li> 使用光束控制软件导入数据收集参数, 该策略计划将提供: i. #969 的起始位置;-旋转轴 ii。#969 的振荡宽度;-每个衍射图像的轴旋转角度 iii。每个衍射图像的曝光时间 iv。完整数据集的图像数 (间接定义总计和 #969;-整个数据采集的轴旋转角度) v. x 射线束衰减的百分比, 以避免过度和辐射损伤 注意: 在我们的机构,运行数据收集时, 自动处理收集的数据的软件管道已经建立好了: (i) 通过 xia2 管道减少 (索引、集成和缩放) 的衍射数据, 从而为用户生成反射 mtz 文件作为分阶段和结构解决方案/细化的输入 48 。(ii) 在数据分析过程中检测到一个重要的反常信号时, 使用 SHELX 的第一个快速自动结构解决方案管道 (fast_ep) 尝试通过实验分期解决重原子下部, 提供了相控电子密度图在可行的情况下。第二个更全面的结构解决方案管道自动承担解决和建立结构的尝试重新使用独立软件套件 49 , 50 , 51 , 52 ;在成功的情况下, 用户将得到一个初始模型和电子密度图。这为用户提供了根据所选择的晶体软件套件完成细化和验证模型的基础. </li>
	</ol>
	</li>
</ol>

作者没有什么可透露的。

在本文中, 我们描述和验证一个综合协议的生物化学和结构表征的碳水化合物 SBPs, 特别强调蛋白质从S. 肺炎链球菌。然而, 这可以作为一个标准的程序来分析其他 SBPs 从不同的有机体, 甚至其他无关的可溶性蛋白质。
该协议的第一部分着重于提供蛋白质稳定性和第四纪结构的生物化学信息, 可用于制备蛋白质样品进行结晶。在热转移分析部分, 我们只描述 pH 和氯化钠浓度的变化, 以保持这个过程的一般性质。尽管如此, 许多其他的缓冲条件可以用类似的方法进行测试, 例如, 包括任何用作稳定添加剂的化合物: 特别是与特定的收缩压结合的实际配体, 在增加 Tm数摄氏度31。在某些情况下, 由于低信号或高荧光背景而导致的变性曲线的定义很差, 这是由蛋白质聚集或部分展开引起的。为了避免这种变化, 一种蛋白质: 可以进行染料滴定来优化不清楚的变性曲线。如果没有得到改善, 筛选各种添加剂, 可以改善蛋白质的稳定性是建议, 和合适的屏幕已经被描述为54。
通常, 大多数 SBPs 是单体在他们的自然环境, 但如图所示, multimerization 可以发生在较高浓度使用的结晶实验, 从而由加工和 SEC 提供的齐聚行为表征重要的是要评估最有利稳定的单分散齐聚状态的结晶。然而, 很难预测在结晶条件中所含的不同化学物质对蛋白质齐聚行为的影响。如果 SEC 和加工检查显示了蛋白质样本的广泛聚集, 我们将建议以下的方法来减少发生这种情况的可能性: 使用新鲜的蛋白质样本 (不冻融) 和扩大热稳定性分析转移化验, 测试可能的添加剂和温和的洗涤剂作为最后的资源, 以尽量减少聚合。本文提出了利用高通量稀疏矩阵商业结晶筛选的基本准则, 以保持该协议的一般性质。然而, 获得高分辨率 X 射线衍射蛋白晶体可能需要迭代微调, 以优化结晶条件的沉淀浓度, pH 值, 添加化学添加剂, 不同的温度,其他影响晶体下落与储层平衡动力学的因素16,17。
该协议的第二部分描述了蛋白质晶体的表征, 以确定 X 射线衍射数据收集的最佳策略, 并特别着重于采集异常数据进行 SAD 分期。即使 SBPs 保持一个类似的一般架构 (有许多沉积的3D 结构可能是可用的作为启动模型), 这些蛋白质的分期的分子置换方法并不总是直接的, 因为变化的二级结构元素和这些蛋白质的内在灵活性。因此, 我们提出可悲的方法, 并强调, 这些蛋白质可能已经有内在的约束金属或事实上的非特异的结合金属从结晶缓冲条件, 这可以提供一系列反常的衍射元素作为我们的常规协议中的标准步骤。
最后, 本协议定义了一个标准的指导工作流程, 以便能够详细描述 SBPs 的生物化学和结构特征, 以提高结构确定成功率, 并加快一般 SBPs 的结构特征。

S. 肺炎球菌) 是一种 gram-positive 细菌, 居住在人类呼吸道上部的症状, 能够迁移到正常的无菌龛位, 导致中耳炎、肺炎、败血症、败血症和脑膜炎1,2。此外, 肺炎球菌感染是导致社区获得性肺炎的主要原因, 它在全球范围内造成了临床和经济负担3,4。抗生素耐药性菌株的S. 肺炎病毒已遍布全球, 虽然七价和十三价的肺炎球菌蛋白共轭疫苗有助于降低抗菌素耐药性率, 取代菌株疫苗的使用已经出现, 并导致对肺炎球菌病新治疗方法的研究的需求增加5,6,7,8。
肺炎球菌依赖于从主机输入的糖类作为碳源9,10;事实上, 它致力于30% 的进口机械的运输32种不同的碳水化合物11,12,13。这些进口商包括至少八 ABC 运输商13。在 ABC 转运器中, 胞外 SBPs 在确定配体特异性方面起着根本性的作用, 并将其呈递给细胞膜上的整体载体以供细胞吸收。SBPs 代表了新疫苗和抗菌素设计的有效目标, 因为它们是表面蛋白及其在细胞过程中的重要作用。
靶蛋白的表征和结构特征的详细描述, 如配体袋和域的灵活性, 为结构药物设计提供了一个有用的工具14,15。x-射线结晶学是选择的方法为蛋白质的结构描述在接近原子决议, 但结晶过程是不可预知的, 费时, 并且不总成功。系统的方法提高了成功率, 重要的因素是样品的质量和稳定性。结晶的成功率受蛋白质化学性质和样品制备方法的影响。它们的作用可以通过生物化学特性16,17进行评估和通知。
一个进一步复杂化为结构设计是晶体阶段问题, 必须解决。随着更多的蛋白质结构变得可用, 许多结构可以通过分子置换的方法来解决, 这需要一个同源结构18。由于 SBPs 提出了一个灵活的领域结构, 分子置换也可能证明挑战19。如果一个结构模型是足够相似的目标蛋白是不可用的, 一些技术可以用来获得实验阶段的20。其中, 单波长反常色散 (SAD) 方法已成为主要技术, 已被广泛用于解决21的相位问题。使用 SAD 方法进一步改进了硬件和软件, 以及数据收集策略, 以便能够检测和使用微弱的异常信号, 以便逐步进行22,23, 24. 此外, 直接解决大分子结构的方法的进展, 这在过去需要衍射数据的原子分辨率, 现在可以利用结合, 例如, 立体知识, 在程序中实施ARCIMOLDO25。泰勒26给出了在晶体学中解决相位问题的方法的一个有用的综述。
在这里, 我们提出了一个合理化的协议的表征碳水化合物运输收缩, SP0092 的S. 肺炎, 集成生化和结构技术 (图 1)。这一 step-by 的协议提供了一个有用的例子测试的策略, 以提高结构研究的成功率, SBPs 一般, 这是在所有王国生活中发现。特别是, 该协议强调了在快速有效的方法中确定蛋白质在溶液中最稳定的聚状态的重要性, 并允许对最佳品种进行结晶实验的鉴定。虽然在蛋白质数据库27中报告了超过500个结构化的收缩组织, 但是分子置换可能具有挑战性, 这是由于两个α/β域的固有弹性性质, 它们由铰链区域19连接。因此, 该协议的第二部分描述了使用 sad 的方法逐步从束缚金属离子, 这是常见的 SBPs, 以及纳入硒和使用硒 (Se) 在 SAD 分期。

<table><tbody><tr><td>SelenoMethionine Medium Complete</td><td>Molecular Dimensions</td><td>MD12-500</td><td>Based on a synthetic M9 minimal media supplemented with glucose, vitamins and amino acids with the exception of L-methionine. Other equivalent products are commercially available by other companies.</td></tr><tr><td>MicroAmp Optical 96-Well plate</td><td>Applied Biosystems</td><td>4306737</td><td>The Applied Biosystems MicroAmp Optical 96-Well Reaction Plate with Barcode is optimized to provide unmatched temperature accuracy and uniformity for fast, efficient PCR amplification. This plate, constructed from a single rigid piece of polypropylene in a 96-well format, is compatible with Applied Biosystems&amp;reg; 96-Well Real-Time PCR systems and thermal cyclers.</td></tr><tr><td>SYPRO Orange</td><td>Molecular Probes</td><td>S6651</td><td>SYPRO Orange Protein Gel Stain is a sensitive, ready-to-use fluorescent stain for proteins in 1D gels. Quite universal and well-established protein dye for hydrophobic regions.</td></tr><tr><td>MicroAmp Optical Adhesive film</td><td>Applied Biosystems</td><td>4311971</td><td>The Applied Biosystems MicroAmp Optical Adhesive Film reduces the chance of well-to-well contamination and sample evaporation when applied to a microplate. It is ideal for optical measurement, because it gives low background.</td></tr><tr><td>7500 Fast Real-time PCR System</td><td>Applied Biosystems</td><td>4362143</td><td>The Applied Biosystems 7500 Fast Real-Time PCR System enables standard 96-well format high speed thermal cycling, significantly reducing your run time for quantitative real-time PCR applications, delivering results in about 30 minutes. The Upgrade Kit is available to upgrade a standard 7500 Real-Time PCR System to the Fast Configuration via a field service installation. Other RT-PCR machine can be used. Data export not easy for the old data analysis software.</td></tr><tr><td>Superdex 200 increase 10/300 GL column</td><td>GE Healthcare</td><td>28990944</td><td>Superdex 200 Increase 10/300 GL is a versatile, prepacked column for size exclusion chromatography in small-scale (mg) preparative purification as well as for characterization and analysis of proteins with molecular weights between 10 000 and 600 000, such as antibodies. Optimal separation ideal for high resolution biophysical techniques.</td></tr><tr><td>DAWN HELEOS II</td><td>Wyatt</td><td></td><td>DAWN HELEOS II is the premier Multi-Angle static Light Scattering (MALS) detector for absolute characterization of the molar mass and size of macromolecules and nanoparticles in solution. The DAWN offers the highest sensitivity, the widest range of molecular weight, size and concentration, and the largest selection of configurations and optional modules for enhanced capabiliites. Other MALS detecting systems from other companies apart from Wyatt have not been tested, so no additional feedback can be provided.</td></tr><tr><td>Superdex 200 5/150 GL</td><td>GE Healthcare</td><td>28906561</td><td>Superdex 200 are prepacked size exclusion chromatography columns for high-resolution small-scale preparative and analytical separations of biomolecules. Superdex 200 has a separation range for molecules with molecular weights between 10 000 and 600 000. The peak separation is not as optimal as for the &quot;increase&quot; version but this model is ideal for the standard day by day use.</td></tr><tr><td>HiLoad 16/600 Superdex 200 pg</td><td>GE Healthcare</td><td>28989335</td><td>HiLoad 16/600 Superdex 200 prep grade are prepacked XK columns designed for high-resolution preparative gel filtration chromatography.</td></tr><tr><td>24 Well &quot;Big&quot; Sitting Drop Crystallization Plate</td><td>MiTeGen</td><td>XQ-P-24S-A</td><td>The 24 well &quot;big&quot; crystallization plate is used mainly for protein crystal screening by sitting drop vapor diffusion techniques, and for crystallization condition optimization. It has quite large reservoir well and sample container, which favor manual handling and big sized protein crystal growth. In addition, its flat surfaces are easily to be sealed by transparent tape or cover slips.</td></tr><tr><td>PCT Pre-Crystallization Test</td><td>Hampton</td><td>HR2-140</td><td>The PCT Pre-Crystallization Test is used to determine the appropriate protein concentration for crystallization screening.</td></tr><tr><td>96 Well CrystalQuick</td><td>Greiner bio-one</td><td>6098xx</td><td>Square-well plates have three crystallization wells per reservoir, making it possible to test 288 samples per plate. Generally used only for the inital screening because their squared edge make crystals fishing difficult.</td></tr><tr><td>Uni-Puck</td><td>Molecular Dimensions</td><td>MD7-601</td><td>The Universal V1-Puck (Uni-puck) is a sample pin storage and shipping container for use with the majority of automated sample mounting systems worldwide - includes the ACTOR, SAM and CATS systems amongst others (Diamond, Soleil, SPring-8, Photon Factory, CLSI and across the USA)</td></tr><tr><td>Standard Foam Dewar</td><td>Molecular Dimensions</td><td>MD7-35</td><td>5.7&quot; diameter by 2.8&quot; deep. 800mL capacity.</td></tr><tr><td>Mounted CryoLoop - 20 micron</td><td>Hampton</td><td>HR4-955</td><td>Mounted CryoLoops with 20 micron diameter nylon. These nylon loops are bonded to hollow, stainless steel MicroTubes&amp;trade; that are used to mount, freeze, and secure the crystal during cryocrystallographic procedures and X-ray data collection. Different sizes exist and they can be adapted in lenght. They are quite versatile tools.</td></tr><tr><td>CryoWand</td><td>Molecular Dimensions</td><td>MD7-411</td><td></td></tr><tr><td>Puck dewar loading tool</td><td>Molecular Dimensions</td><td>MD7-607</td><td>This tool is used to separate uni-pucks to load them into the robot dewar. It consists of two pieces: a Teflon tube part and a metal rod part.</td></tr></tbody></table>

biochemical and structural characterization of the carbohydrate transport substrate-binding-protein sp0092

为防止多重耐药性菌株的迅速增加, 必须研制新的抗菌素和疫苗, 以防止肺炎链球菌的。碳水化合物基质结合蛋白 (SBPs) 是发展蛋白质疫苗和新抗菌素的可行目标, 因为它们的胞外定位和对肺炎球菌代谢的碳水化合物进口的中心地位,分别.这里描述的是一个合理化的综合协议, 对 SP0092 进行全面的表征, 可以扩展到其他碳水化合物 SBPs 从肺炎球菌和其他细菌。这一过程可以帮助结构设计这类蛋白质的抑制剂。本文的第一部分介绍了热位移法、多角度光散射 (加工) 和尺寸排斥色谱 (SEC) 的生化分析协议, 该方法优化了样品的稳定性和均匀性, 以结晶试验, 从而提高了成功的几率。本程序的第二部分描述了使用可调谐波长的反常衍射同步光束, 以及可用于解析结晶蛋白的数据的数据收集协议。结构.

这一综合协议已被证明是成功的四 (两个出版和两个未发表的结构) 的六碳水化合物结合蛋白目标从肺炎球菌分析到日期32,53。在本节中, 我们介绍了 SP0092 的生物化学和结构特征, 作为指导 SBPs 结构研究的典型结果。
在 SP0092 已被表达和纯化为先前定义的32后, 用热漂移法分析了纯化蛋白的缓冲稳定性: SP0092 在 pH 值6.5 和氯化钠的存在下, 在 0-0.2 m 中呈现增加的 Tm浓度范围 (图 3A)。因此, 以下步骤的缓冲解决方案被定义为: 0.02 m MES pH 6.5、0.2 m 氯化钠、2.5% (v/v) 甘油、0.5 mM TCEP。SP0092 的不同齐聚态的绝对摩尔质量是由加工耦合到 SEC 测量187.2、140.8、97.0 和 49.4 kDa 的分子量, 分别对应于 tetrameric、三、二和单体物种 (图 3B)。对不同蛋白稀释的 SEC 剖面的分析表明, 齐聚物是由增加的蛋白质浓度引发的, 这表明更大的寡聚物在较高的浓度下比通常使用的更稳定。结晶.事实上, 被纯化的较大的聚物种的结晶试验, 成功地产生了蛋白质晶体, 而单体物种没有。
用 X 射线衍射法表征了 SP0092 的天然和硒-蛋氨酸标记形式的优化晶体。这些晶体中的 X 射线荧光的测量结果显示, 在两种情况下, 锌与蛋白质结合的发射峰, 而硒只被发现的硒-蛋氨酸晶体的预期。随后, 在硒和锌边缘进行 x 射线吸收扫描, 提供了直接的实验数据, 将入射 x 射线波长调整到晶体中锌或硒的各自 x 射线吸收边缘, 使异常信号达到最大。可从结果数据中获得 (图 4A-B)。
在低传输测量三衍射模式后, 利用埃德娜 (图 4C) 所建议的数据收集策略获得了一个完整的异常数据集。异常信号的出现触发自动分阶段管道在光束, 以确定结构, 并根据最初的实验阶段得出, 产生初始地图和模型, 然后可以提炼和验证 (图4D).
总之, 技术的潜在缺陷主要集中在晶体的可用性和质量上。优化的缓冲条件, 以提高蛋白质的稳定性, 以及确定最合适的聚状态的蛋白质使用, 当多个齐聚物被确定在溶液中, 可以减少失效的风险在结晶阶段.利用早期发现的束缚金属离子, 可以加快结构的解决, 避免在分子置换方法失败时不必要的硒标记蛋白的产生。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56294/56294fig1.jpg"> 
图1。碳水化合物 SBPs 的生物化学和结构表征工作流程图.<a href="//ecsource.jove.com/files/ftp_upload/56294/56294fig1large.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/56294/56294fig2.jpg"> 
图2。GDA 中的晶体特性.(A) GDA 光束控制软件的 X 射线荧光控制标签的屏幕截图。(B) GDA 中 X 射线能量边缘扫描控制卡的屏幕截图。(C) GDA 中的数据收集水晶筛选选项卡的屏幕快照。<a href="//ecsource.jove.com/files/ftp_upload/56294/56294fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56294/56294fig3.jpg"> 
图3。SP0092 的生物化学特性39-491.(A) 3 维曲面图, 它将 SP009239-491的熔化温度绘制为缓冲液的 pH 和氯化钠浓度的函数。(B) SEC 和加工结果为 SP009239-491。在 280 nm 的吸收以蓝色显示。不同齐聚状态的摩尔质量以红色显示。面板 (B) 已从32中修改。<a href="//ecsource.jove.com/files/ftp_upload/56294/56294fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56294/56294fig4.jpg"> 
图4。SP009239-491的结构特征.(A) 和 (B) X 射线吸收能量扫描在 SP009239-491晶体的锌和硒边缘。从含锌和硒的晶体中测定的荧光值分别显示为蓝色和青色, 计算出的 f "和 f" 异常散射分数为绿色和红色。(C) 从 SP009239-491晶体中收集的 X 射线衍射图案的示例。(D) SP009239-491二聚体结构的卡通表示。一个 protomer 是有色白色和其他洋红色 (残余 39-366) 和紫罗兰 (残余 367-491), 和反常的散射原子分别显示为蓝色和青色球锌和硒。<a href="//ecsource.jove.com/files/ftp_upload/56294/56294fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>0.1 M 柠檬酸 pH 值4。0</td>
			<td>0.1 M 柠檬酸 pH 值4。5</td>
			<td>0.1 米磷酸酯 pH 值5。0</td>
			<td>0.1 米柠檬酸盐 pH 值5。5</td>
			<td>0.1 M 双三酸6</td>
			<td>0.1 M ADA pH 6。5</td>
			<td>0.1 M 拖把 pH 7</td>
			<td>0.1 M HEPES pH 7。5</td>
			<td>0.1 M 咪唑 pH 值8。0</td>
			<td>0.1 M 三 pH 8。5</td>
			<td>0.1 M 棋 pH 9</td>
			<td>0.1 M 棋 pH 9。5</td>
		</tr>
		<tr>
			<td>氯化钠 0 M</td>
			<td>40μ l</td>
			<td>40μ l</td>
			<td>40μ l</td>
			<td>40μ l</td>
			<td>40μ l</td>
			<td>40μ l</td>
			<td>40μ l</td>
			<td>40μ l</td>
			<td>40μ l</td>
</tr></tbody></table>			<td>40μ l</td>
			<td>40μ l</td>
			<td>40μ l</td>
		
		<tr>
			<td>氯化钠 0.1 M</td>
			<td>40μ l</td>
			<td>40μ l</td>
			<td>40μ l</td>
			<td>40μ l</td>
			<td>40μ l</td>
			<td>40μ l</td>
			<td>40μ l</td>
			<td>40μ l</td>
			<td>40μ l</td>
			<td>40μ l</td>
			<td>40μ l</td>
			<td>40μ l</td>
		</tr>
		<tr>
			<td>氯化钠 0.2 M</td>
			<td>40μ l</td>
			<td>40μ l</td>
			<td>40μ l</td>
			<td>40μ l</td>
			<td>40μ l</td>
			<td>40μ l</td>
			<td>40μ l</td>
			<td>40μ l</td>
			<td>40μ l</td>
			<td>40μ l</td>
			<td>40μ l</td>
			<td>40μ l</td>
		</tr>
		<tr>
			<td>氯化钠 0.5 M</td>
			<td>40μ l</td>
			<td>40μ l</td>
			<td>40μ l</td>
			<td>40μ l</td>
			<td>40μ l</td>
			<td>40μ l</td>
			<td>40μ l</td>
			<td>40μ l</td>
			<td>40μ l</td>
			<td>40μ l</td>
			<td>40μ l</td>
			<td>40μ l</td>
		</tr>
	

表1。热位移测定的缓冲成分。

Watch this Scientific Journal Video about Biochemical and Structural Characterization of the Carbohydrate Transport Substrate-binding-protein SP0092 at JoVE.com

Biochemical and Structural Characterization of the Carbohydrate Transport Substrate-binding-protein SP0092

一个简化的协议, 以执行广泛的生物化学和结构表征的碳水化合物基质结合蛋白从肺炎链球菌。

碳水化合物转运基质结合蛋白 SP0092 的生化和结构表征

Development of new antimicrobials and vaccines for Streptococcus pneumoniae (pneumococcus) are necessary to halt the rapid rise in multiple resistant ...

biochemical-structural-characterization-carbohydrate-transport

Research

JoVE Journal

Biochemistry

29.8K 次观看.  Harwell Science & Innovation Campus. 一个简化的协议, 以执行广泛的生物化学和结构表征的碳水化合物基质结合蛋白从肺炎链球菌。

视频: Biochemical and Structural Characterization of the Carbohydrate Transport Substrate-binding-protein SP0092

Characterization of Glycoproteins with the Immunoglobulin Fold by X-Ray Crystallography and Biophysical Techniques

Glycoproteins on the surface of cells play critical roles in cellular function, including signalling, adhesion and transport. On leukocytes, several of these glycoproteins possess immunoglobulin (Ig) folds and are central to immune recognition and regulation. Here, we present a platform for the design, expression and biophysical characterization of the extracellular domain of human B cell receptor CD22. We propose that these approaches are broadly applicable to the characterization of mammalian glycoprotein ectodomains containing Ig domains. Two suspension human embryonic kidney (HEK) cell lines, HEK293F and HEK293S, are used to express glycoproteins harbouring complex and high-mannose glycans, respectively. These recombinant glycoproteins with different glycoforms allow investigating the effect of glycan size and composition on ligand binding. We discuss protocols for studying the kinetics and thermodynamics of glycoprotein binding to biologically relevant ligands and therapeutic antibody candidates. Recombinant glycoproteins produced in HEK293S cells are amenable to crystallization due to glycan homogeneity, reduced flexibility and susceptibility to endoglycosidase H treatment. We present methods for soaking glycoprotein crystals with heavy atoms and small molecules for phase determination and analysis of ligand binding, respectively. The experimental protocols discussed here hold promise for the characterization of mammalian glycoproteins to give insight into their function and investigate the mechanism of action of therapeutics.

We present approaches for the biophysical and structural characterization of glycoproteins with the immunoglobulin fold by biolayer interferometry, isothermal titration calorimetry, and X-ray crystallography.

用 X 射线晶体学和生物物理技术表征免疫球蛋白折叠的糖蛋白

Glycoproteins on the surface of cells play critical roles in cellular function, including signalling, adhesion and transport. On leukocytes, several of ...

科研

生物化学

Spectrophotometric Methods for the Study of Eukaryotic Glycogen Metabolism

Glycogen is synthesized as a storage form of glucose by a wide array of organisms, ranging from bacteria to animals. The molecule comprises linear chains of &#945;1,4-linked glucose residues with branches introduced through the addition of &#945;1,6-linkages. Understanding how the synthesis and degradation of glycogen are regulated and how glycogen attains its characteristic branched structure requires the study of the enzymes of glycogen storage. However, the methods most commonly used to study these enzyme activities typically employ reagents or techniques that are not available to all investigators. Here, we discuss a battery of procedures that are technically simple, cost-effective, and yet still capable of providing valuable insight into the control of glycogen storage. The techniques require access to a spectrophotometer, operating in the range of 330 to 800 nm, and are described assuming that the users will employ disposable, plastic cuvettes. However, the procedures are readily scalable and can be modified for use in a microplate reader, allowing highly parallel analysis.

Techniques to measure the activity of key enzymes of glycogen metabolism are presented, using a simple spectrophotometer operating in the visible range.

用于研究真核糖原代谢的分光光度法

Glycogen is synthesized as a storage form of glucose by a wide array of organisms, ranging from bacteria to animals. The molecule comprises linear chains ...

The Application of Open Searching-based Approaches for the Identification of Acinetobacter baumannii O-linked Glycopeptides

Protein glycosylation is increasingly recognized as a common modification within bacterial organisms, contributing to prokaryotic physiology and optimal infectivity of pathogenic species. Due to this, there is increasing interest in characterizing bacterial glycosylation and a need for high-throughput analytical tools to identify these events. Although bottom-up proteomics readily enables the generation of rich glycopeptide data, the breadth and diversity of glycans observed in prokaryotic species make the identification of bacterial glycosylation events extremely challenging.
Traditionally, the manual determination of glycan compositions within bacterial proteomic datasets made this a largely bespoke analysis restricted to field-specific experts. Recently, open searching-based approaches have emerged as a powerful alternative for the identification of unknown modifications. By analyzing the frequency of unique modifications observed on peptide sequences, open searching techniques allow the identification of common glycans attached to peptides within complex samples. This article presents a streamlined workflow for the interpretation and analysis of glycoproteomic data, demonstrating how open searching techniques can be used to identify bacterial glycopeptides without prior knowledge of the glycan compositions.
Using this approach, glycopeptides within samples can rapidly be identified to understand glycosylation differences. Using Acinetobacter baumannii as a model, these approaches enable the comparison of glycan compositions between strains and the identification of novel glycoproteins. Taken together, this work demonstrates the versatility of open database-searching techniques for the identification of bacterial glycosylation, making the characterization of these highly diverse glycoproteomes easier than ever before.

The Application of Open Searching-based Approaches for the Identification of Acinetobacter baumannii O-linked Glycopeptides

Open searching enables the identification of glycopeptides decorated with previously unknown glycan compositions. Within this article, a streamlined approach for undertaking open searching and subsequent glycan-focused glycopeptide searches are presented for bacterial samples using Acinetobacter baumannii as a model.

基于开放搜索的方法在 鲍曼不动杆菌 O-连锁糖肽鉴定中的应用

Protein glycosylation is increasingly recognized as a common modification within bacterial organisms, contributing to prokaryotic physiology and optimal ...

Concanavalin A-Based Sedimentation Assay to Measure Substrate Binding of Glucan Phosphatases

Glucan phosphatases belong to the larger family of dual specificity phosphatases (DSP) that dephosphorylate glucan substrates, such as glycogen in animals and starch in plants. The crystal structures of glucan phosphatase with model glucan substrates reveal distinct glucan-binding interfaces made of DSP and carbohydrate-binding domains. However, quantitative measurements of glucan-glucan phosphatase interactions with physiologically relevant substrates are fundamental to the biological understanding of the glucan phosphatase family of enzymes and the regulation of energy metabolism. This manuscript reports a Concanavalin A (ConA)-based in vitro sedimentation assay designed to detect the substrate binding affinity of glucan phosphatases against different glucan substrates.&#160;As a proof of concept, the dissociation constant (KD) of glucan phosphatase Arabidopsis thaliana Starch Excess4 (SEX4) and amylopectin was determined. The characterization of SEX4 mutants and other members of the glucan phosphatase family of enzymes further demonstrates the utility of this assay to assess the differential binding of protein- carbohydrate interactions. These data demonstrate the suitability of this assay to characterize a wide range of starch and glycogen interacting proteins.

This method describes a lectin-based in vitro sedimentation assay to quantify the binding affinity of glucan phosphatase and amylopectin. This co-sedimentation assay is reliable for measuring glucan phosphatase substrate binding and can be applied to various solubilized glucan substrates.

基于刀豆球蛋白A的沉降测定法，用于测量葡聚糖磷酸酶的底物结合

Glucan phosphatases belong to the larger family of dual specificity phosphatases (DSP) that dephosphorylate glucan substrates, such as glycogen in animals ...

Engineering Antiviral Agents via Surface Plasmon Resonance

Surface plasmon resonance (SPR) is used to measure hemagglutinin (HA) binding to domain-swapped Cyanovirin-N (CV-N) dimer and to monitor interactions between mannosylated peptides and CV-N's high-affinity binding site. Virus envelope spikes gp120, HA, and Ebola glycoprotein (GP) 1,2 have been reported to bind both high- and low-affinity binding sites on dimeric CVN2. Dimannosylated HA peptide is also bound at the two low-affinity binding sites to an engineered molecule of CVN2, which is bearing a high-affinity site for the respective ligand and mutated to replace a stabilizing disulfide bond in the carbohydrate-binding pocket, thus confirming multivalent binding. HA binding is shown to one high-affinity binding site of pseudo-antibody CVN2 at a dissociation constant (KD) of 275 nM that further neutralizes human immunodeficiency virus type 1 (HIV-1) through oligomerization. Correlating the number of disulfide bridges in domain-swapped CVN2, which are decreased from 4 to 2 by substituting cystines into polar residue pairs of glutamic acid and arginine, results in reduced binding affinity to HA. Among the strongest interactions, Ebola GP1,2 is bound by CVN2 with two high-affinity binding sites in the lower nanomolar range using the envelope glycan without a transmembrane domain. In the present study, binding of the multispecific monomeric CV-N to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) glycoprotein is measured at KD = 18.6 &#181;M as compared with nanomolar KD to those other virus spikes, and via its receptor-binding domain in the mid-&#181;-molar range.

Engineering Antiviral Agents via Surface Plasmon Resonance

The present protocol describes new tools for SPR binding assays to examine CV-N binding to HA, S glycoprotein, related hybrid-type glycans, and high-mannose oligosaccharides. SPR is used to determine the KD for binding either dimeric or monomeric CV-N to these glycans.

通过表面等离子体共振 设计 抗病毒药物

Surface plasmon resonance (SPR) is used to measure hemagglutinin (HA) binding to domain-swapped Cyanovirin-N (CV-N) dimer and to monitor interactions ...

生物工程

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that currently limits the average life expectancy of sufferers to &#60;40 years of age. The development of novel drug molecules to restore the activity of CFTR is an important goal in the treatment CF, and the isolation of functionally active CFTR is a useful step towards achieving this goal.
We describe two methods for the purification of CFTR from a eukaryotic heterologous expression system, S. cerevisiae. Like prokaryotic systems, S. cerevisiae can be rapidly grown in the lab at low cost, but can also traffic and posttranslationally modify large membrane proteins. The selection of detergents for solubilization and purification is a critical step in the purification of any membrane protein. Having screened for the solubility of CFTR in several detergents, we have chosen two contrasting detergents for use in the purification that allow the final CFTR preparation to be tailored to the subsequently planned experiments.
In this method, we provide comparison of the purification of CFTR in dodecyl-&#946;-D-maltoside (DDM) and 1-tetradecanoyl-sn-glycero-3-phospho-(1&#39;-rac-glycerol) (LPG-14). Protein purified in DDM by this method shows ATPase activity in functional assays. Protein purified in LPG-14 shows high purity and yield, can be employed to study post-translational modifications, and can be used for structural methods such as small-angle X-ray scattering and electron microscopy. However it displays significantly lower ATPase activity.

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Heterologous expression and purification of the cystic fibrosis transmembrane conductance regulator (CFTR) are significant challenges and limiting factors in the development of drug therapies for cystic fibrosis. This protocol describes two methods for the isolation of milligram quantities of CFTR suitable for functional and structural studies.&#160;

囊性纤维化跨膜电导调节蛋白表达纯化<em&gt;酿酒酵母</em

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that ...

生物学

Real Time Measurements of Membrane Protein:Receptor Interactions Using Surface Plasmon Resonance (SPR)

Protein-protein interactions are pivotal to most, if not all, physiological processes, and understanding the nature of such interactions is a central step in biological research. Surface Plasmon Resonance (SPR) is a sensitive detection technique for label-free study of bio-molecular interactions in real time. In a typical SPR experiment, one component (usually a protein, termed 'ligand') is immobilized onto a sensor chip surface, while the other (the 'analyte') is free in solution and is injected over the surface. Association and dissociation of the analyte from the ligand are measured and plotted in real time on a graph called a sensogram, from which pre-equilibrium and equilibrium data is derived. Being label-free, consuming low amounts of material, and providing pre-equilibrium kinetic data, often makes SPR the method of choice when studying dynamics of protein interactions. However, one has to keep in mind that due to the method's high sensitivity, the data obtained needs to be carefully analyzed, and supported by other biochemical methods. 
SPR is particularly suitable for studying membrane proteins since it consumes small amounts of purified material, and is compatible with lipids and detergents. This protocol describes an SPR experiment characterizing the kinetic properties of the interaction between a membrane protein (an ABC transporter) and a soluble protein (the transporter's cognate substrate binding protein).

Real Time Measurements of Membrane Protein:Receptor Interactions Using Surface Plasmon Resonance SPR

Surface Plasmon Resonance (SPR) is a label-free method for detecting bio-molecular interactions in real time. Herein, a protocol for a membrane protein:receptor interaction experiment is described, while discussing the pros and cons of the technique.

膜蛋白的实时测量:受体相互作用使用表面等离子体共振（SPR）

Protein-protein interactions are pivotal to most, if not all, physiological processes, and  understanding the nature of such interactions is a central ...

Automated Modular High Throughput Exopolysaccharide Screening Platform Coupled with Highly Sensitive Carbohydrate Fingerprint Analysis

Many microorganisms are capable of producing and secreting exopolysaccharides (EPS), which have important implications in medical fields, food applications or in the replacement of petro-based chemicals. We describe an analytical platform to be automated on a liquid handling system that allows the fast and reliable analysis of the type and the amount of EPS produced by microorganisms. It enables the user to identify novel natural microbial exopolysaccharide producers and to analyze the carbohydrate fingerprint of the corresponding polymers within one day in high-throughput (HT). Using this platform, strain collections as well as libraries of strain variants that might be obtained in engineering approaches can be screened. The platform has a modular setup, which allows a separation of the protocol into two major parts. First, there is an automated screening system, which combines different polysaccharide detection modules: a semi-quantitative analysis of viscosity formation via a centrifugation step, an analysis of polymer formation via alcohol precipitation and the determination of the total carbohydrate content via a phenol-sulfuric-acid transformation. Here, it is possible to screen up to 384 strains per run. The second part provides a detailed monosaccharide analysis for all the selected EPS producers identified in the first part by combining two essential modules: the analysis of the complete monomer composition via ultra-high performance liquid chromatography coupled with ultra violet and electrospray ionization ion trap detection (UHPLC-UV-ESI-MS) and the determination of pyruvate as a polymer substituent (presence of pyruvate ketal) via enzymatic oxidation that is coupled to a color formation. All the analytical modules of this screening platform can be combined in different ways and adjusted to individual requirements. Additionally, they can all be handled manually or performed with a liquid handling system. Thereby, the screening platform enables a huge flexibility in order to identify various EPS.

We present an automated modular high-throughput-method for the identification and characterization of microbial exopolysaccharides in small scale. This method combines a fast preselection to analyze the total amount of secreted polysaccharides with a detailed carbohydrate fingerprint to enable the fast screening of newly isolated bacterial strains or entire strain collections.

再加上高灵敏度指纹碳水化合物自动分析模块化高通量筛选胞外多糖平台

Many microorganisms are capable of producing and secreting exopolysaccharides (EPS), which have important implications in medical fields, food ...

Demonstration of Heterologous Complexes formed by Golgi-Resident Type III Membrane Proteins using Split Luciferase Complementation Assay

The goal of this protocol is to explore the applicability of the most recent variant of split luciferase complementation for demonstrating heterologous complexes formed by nucleotide sugar transporters (NSTs). These ER- and Golgi-resident multitransmembrane proteins carry the cytoplasmically synthesized nucleotide sugars across organelle membranes to supply enzymes that mediate glycosylation with their substrates. NSTs exist as dimers and/or higher oligomers. Heterologous interactions between&#160;different NSTs have also been reported. To verify whether the technique is suitable for studying the phenomenon of NST heteromerization, we tested it against a combination of the two Golgi-resident NSTs that have been previously shown to associate by several other means. The&#160;luciferase complementation assay appears to be particularly suitable for studying interactions between Golgi-resident membrane proteins, as it does not require high expression levels, which often trigger protein mislocalization and increase the risk of false positives.

The&#160;protocol presented here is intended to demonstrate the occurrence of heterologous interactions between Golgi-resident type III membrane proteins with cytoplasmically exposed N- and/or C-termini in live mammalian cells using the most recent variant of the split luciferase complementation assay.

使用分裂荧光素酶互补测定法展示高尔基体驻留III型膜蛋白形成的异源复合物

The goal of this protocol is to explore the applicability of the most recent variant of split luciferase complementation for demonstrating heterologous ...

Small-Scale Plasma Membrane Preparation for the Analysis of Candida albicans Cdr1-mGFPHis

The successful biochemical and biophysical characterization of ABC transporters depends heavily on the choice of the heterologous expression system. Over the past two decades, we have developed a yeast membrane protein expression platform that has been used to study many important fungal membrane proteins. The expression host Saccharomyces cerevisiae AD&#916;&#916; is deleted in seven major endogenous ABC transporters and it contains the transcription factor Pdr1-3 with a gain-of-function mutation that enables the constitutive overexpression of heterologous membrane protein genes stably integrated as single copies at the genomic PDR5 locus. The creation of versatile plasmid vectors and the optimization of one-step cloning strategies enables the rapid and accurate cloning, mutagenesis, and expression of heterologous ABC transporters. Here, we describe the development and use of a novel protease-cleavable mGFPHis double tag (i.e., the monomeric yeast enhanced green fluorescent protein yEGFP3 fused to a six-histidine affinity purification tag) that was designed to avoid possible interference of the tag with the protein of interest and to increase the binding efficiency of the His tag to nickel-affinity resins. The fusion of mGFPHis to the membrane protein ORF (open reading frame) enables easy quantification of the protein by inspection of polyacrylamide gels and detection of degradation products retaining the mGFPHis tag. We demonstrate how this feature facilitates detergent screening for membrane protein solubilization. A protocol for the efficient, fast, and reliable isolation of the small-scale plasma membrane preparations of the C-terminally tagged Candida albicans multidrug efflux transporter Cdr1 overexpressed in S. cerevisiae AD&#916;&#916;, is presented. This small-scale plasma membrane isolation protocol generates high-quality plasma membranes within a single working day. The plasma membrane preparations can be used to determine the enzyme activities of Cdr1 and Cdr1 mutant variants.

Small-Scale Plasma Membrane Preparation for the Analysis of Candida albicans Cdr1-mGFPHis

This article presents a small-scale plasma membrane isolation protocol for the characterization of Candida albicans ABC (ATP-binding cassette) protein Cdr1, overexpressed in Saccharomyces cerevisiae. A protease-cleavable C-terminal mGFPHis double tag with a 16-residue linker between Cdr1 and the tag was designed to facilitate the purification and detergent-screening of Cdr1.

用于分析 念珠菌 Cdr1-mGFPHis分析的小尺度等离子膜制备

The successful biochemical and biophysical characterization of ABC transporters depends heavily on the choice of the heterologous expression system. Over ...

碳水化合物转运基质结合蛋白 SP0092 的生化和结构表征

摘要

探索更多视频

此视频中的章节

用 X 射线晶体学和生物物理技术表征免疫球蛋白折叠的糖蛋白

用于研究真核糖原代谢的分光光度法

基于开放搜索的方法在鲍曼不动杆菌 O-连锁糖肽鉴定中的应用

基于刀豆球蛋白A的沉降测定法，用于测量葡聚糖磷酸酶的底物结合

通过表面等离子体共振设计抗病毒药物

囊性纤维化跨膜电导调节蛋白表达纯化

膜蛋白的实时测量:受体相互作用使用表面等离子体共振（SPR）

再加上高灵敏度指纹碳水化合物自动分析模块化高通量筛选胞外多糖平台

使用分裂荧光素酶互补测定法展示高尔基体驻留III型膜蛋白形成的异源复合物

用于分析念珠菌 Cdr1-mGFPHis分析的小尺度等离子膜制备

碳水化合物转运基质结合蛋白 SP0092 的生化和结构表征

摘要

探索更多视频

此视频中的章节

用 X 射线晶体学和生物物理技术表征免疫球蛋白折叠的糖蛋白

用于研究真核糖原代谢的分光光度法

基于开放搜索的方法在 鲍曼不动杆菌 O-连锁糖肽鉴定中的应用

基于刀豆球蛋白A的沉降测定法，用于测量葡聚糖磷酸酶的底物结合

通过表面等离子体共振 设计 抗病毒药物

囊性纤维化跨膜电导调节蛋白表达纯化

膜蛋白的实时测量:受体相互作用使用表面等离子体共振（SPR）

再加上高灵敏度指纹碳水化合物自动分析模块化高通量筛选胞外多糖平台

使用分裂荧光素酶互补测定法展示高尔基体驻留III型膜蛋白形成的异源复合物

用于分析 念珠菌 Cdr1-mGFPHis分析的小尺度等离子膜制备

基于开放搜索的方法在鲍曼不动杆菌 O-连锁糖肽鉴定中的应用

通过表面等离子体共振设计抗病毒药物

用于分析念珠菌 Cdr1-mGFPHis分析的小尺度等离子膜制备