作者要感谢美国陆军研究实验室 (ARL) 博士后奖学金计划的支持, 由橡树岭联合大学管理, 通过与 ARL 的合同, 通过任命 Dr. 贾斯汀 p. Jahnke。剩余资金由 ARL 的传感器和电子设备局提供。作者还要感谢多尔蒂实验室在 UCSB 共享细菌显示库和信息克隆的 YPet mona 试剂, Dr. 布林亚当斯支持克隆 YPet 蒙娜丽莎试剂在 ARL, Dr. 约书亚 Kogot 为他的初步工作和在细菌显示库筛选的训练, 阿丽娜伍德化学和生物中心的平静, 用于表达和纯化 abrax 和 rivax 蛋白的质粒, 用于分离 PA 结合肽的技术支持, 秦郭为 abrax 结合肽的初步实验提供技术支持, Dr. 洁西卡特雷尔为有关此手稿的有益讨论。

作者没有透露

筛选细菌显示库是一种成功的方法隔离亲和试剂, 和多肽提供了一个有用的替代抗体的识别时, 特定的标准, 如在极端环境的稳定性。这些库的筛选已通过这里描述的 autoMCS 方法进行了简化, 而商业上可用的 autoMCS 平台将此技术扩展到更广泛的用户。与传统的细菌显示库的交替式 MCS/外地资产管制分类方法相比, autoMCS 方法的费用较低, 因为它们不需要投资于一个更昂贵的、能够对绑定单元进行分类和隔离的外地机构。而不是这里使用的流式细胞仪的类型, 它只能够分析14。autoMCS 成功筛选的关键步骤包括分离方案选择, 对潜在的交叉反应物进行负排序, 以减少误报, 利用外地资产管理系统监测图书馆的丰富性, 以确定何时停止筛选, 以及对候选池进行智能分析, 以避免对数百名候选者进行繁琐的筛选。
本文中的例子说明了选择的细菌显示库的成功筛选两个单独的目标, PA 和 abrax, 虽然稍有不同的分析策略, 由于不同的肽序列为每个池候选人经过四轮的筛选。PA 是更直接的情况下, 序列分析显示明确的趋势, 从多肽序列, 重复至少一次在144殖民地。仅此一项就足以确定 pa 目标的协商一致序列, 而那些包含共识或类似序列的殖民地是在绑定到 pa 与绑定到亲和负控制的比率方面的最佳候选者。然而, 情况并非总是如此。对于 abrax 目标, 测序100菌落导致五重复序列, 但这些序列中的趋势是不太清楚, 除了倾向于含有芳香氨基酸残留物和天门冬氨酸或胺。从重复序列中所预测的 "共识" 只能产生和测试与 abrax 的亲和性, 但由于没有父序列包含精确的一致序列, 我们返回到序列化的菌落列表, 并观察到其他有更多共同倾向的候选人。事实上, 两个殖民地包含了一个类似的序列的 "共识" 从单独的重复, (FWDTWF 而不是 FWAWF), 这有相同的趋势, 色氨酸, 苯丙胺, 和天门冬氨酸残留, 但有不同的间距。其中一个肽是重复序列, 一个不是。这两个序列, 连同第三个序列包含一个类似的变体, 是在前5的粘合剂测试的亲和力的 abrax。顶部粘合剂整体, AX-A05, 也显示相似的趋向。abrax 的结果表明, 根据所选择的目标, 有时需要在序列分析与亲和性和特异性分析之间进行多次交替的方法。abrax 目标的结果还显示了在非常相似的蛋白质 (rivax 1,15) 上可以实现的特异性级别。
为我们的研究选择的 autoMCS 程序是 Posselds 和 Posseld, 这都是为积极选择的标记目标细胞低频, 少于5% 的初始人口。在这两种情况下, 细胞通过两个磁性柱。但是, 在 Posselds 程序的情况下, 单元格通过第一列的速度更慢, 从而增加曝光时间41。除了商用 autoMCS 设备制造商提供的程序说明 (请参见材料表) 41, 这些程序是在对几个潜在程序进行初步比较后选择的。每个程序的能力, 以避免从一个同质的负面控制细胞培养假阳性, 并成功地将已知的粘结剂从含有负控制细胞的混合培养中分离出来, 以减少比较已知粘合剂的百分比。如果与此处描述的应用程序有明显的偏差, 类似的比较可能有助于新应用程序的程序选择。然而, 以前公布的结果比较这两种程序 (Posseld 和 Posselds) 分离肽亲和试剂的 PA 表明, 使用这些程序的所有四轮筛选导致的多肽的分离与类似PA 的亲和性和特异性以及 WXCFTC 共识的确定。主要区别是 Posseld, 以它的快速的流速, 排序更加快速地和结合的亲和力为目标是更高的, 因此在仅二个轮筛选之后, 而使用 Posselds 导致了更加独特的序列在四轮以后筛选14. 从这一点中学习, 当筛选 abrax 结合肽以合并两种好处时, 策略略有变化: Posselds 用于1轮, 允许在这一关键步骤中有更多的曝光时间, 其中的多样性最高, 但随后在 2-4 1,15中, 程序被切换到 Posseld 以更快地隔离最高的亲和力活页夹。这似乎是理想的 abrax, 特别是因为 nMFI 和百分比绑定都更高的 abrax 排序轮4相比, PA 分类轮4与任何程序 (图 2和 图 3和 Sarkes et al. 201514), 但要进行更具决定性的比较, 则需要使用一种策略与同一目标进行直接比较。哪个程序最适合特定的应用程序可能取决于单个目标、所使用的排序库以及在此处描述的条件发生任何更改时所达到的多肽表达式的级别。
没有讨论这里是潜在的结果从筛选与非生物材料, 但我们也使用了相同的细菌显示库的筛选反对散装铝2。这项研究没有使用 autoMCS, 但类似的研究可以完成使用 autoMCS 如果材料是可用的, 或可以涂上或共轭, 磁珠。在大块铝研究中, 个体氨基酸分析和二级结构建模有助于了解什么是驱动的结合亲和性的分离肽, 因为没有共识序列确定2。即使有了生物目标, 这可能需要了解什么是驱动绑定亲和力的一些目标, 这就是为什么电子表格生成的 "eCPX_Sequencing" 宏包括一个标签与个别残留频率分析。如果除了缺乏共识外, 没有出现重复序列, 而且剩余频率没有显示任何模式, 则可能需要其他类型的分析。此外, 可能还需要进一步的分类, 以避免筛选出更多的候选者, 以便选择那些与预期目标有足够亲和力的候选人。
随着下一代测序的增加, 可以进行更深入的研究, 以确定最有希望的候选者, 然后再测试亲和性, 并确定每轮筛选后的富集程度。但是, 如果这些序列没有足够高的频率以在数十个或数以百计的蚁群中进行常规的菌落测序, 那么移动到绑定分析步骤的顺序可能需要由克隆来重新创建。随着这项技术的进步, 变得越来越不昂贵和更常规, 特别是有一个选择的殖民地隔离, 这将可能是最好的方式来分析筛选数据。它可能会导致解释的方式个别序列, 和整个图书馆, 演变。此外, 分类库中的细菌可能随着时间的推移而变异, 这可能会对细胞进行分类, 使其具有更快的生长速率或细菌, 过度基因组蛋白能够结合材料, 即使没有显示支架和肽表达,实例.因此, 一旦有希望的候选者出现, 就值得分离质粒 DNA, retransforming 新鲜的大肠杆菌,和通过外地资产管制组确认多肽结合。如果计划使用的肽在一个无细胞的格式, 亲和性也应评估的自由肽。例如, 通过对靶点的细菌显示进行多肽亲和试剂的合成, 并采用酶联 immunosorbant 法 (ELISA) 和细胞 (通过) 和离体细胞等传统方法进行分析。(通过 ELISA) 亲和性的比较使用的 Kd由两种方法确定的7。
虽然生物素-亲和相互作用的强度使它成为捕获蛋白质靶和结合肽的理想策略, 但此过程可以被修改以获取其他捕获策略。直接耦合蛋白质的磁性珠子, 如环氧树脂和羧酸珠, 已经成功, 并取消了一个结合的步骤。但是, 根据附件策略, 直接附件可能会以特定或非特定的方式阻碍潜在的绑定站点。一个额外的优势, 使用亲和珠是不稳定的蛋白质目标可以新鲜化在30分钟或储存冷冻, 如果需要, 而珠子本身往往需要更长的孵化与蛋白质的交叉连接, 并一般存储在 2-c。建议的化蛋白靶的起始浓度在这里, 600 nM, 已经成功的多个目标, 但它可能是可能的分离肽捕获试剂增加亲和力, 如果这种浓度降低。此外, 该方法还可以扩展到其他细菌显示库和其他生物体。例如, 这些步骤可以在没有氧气的情况下进行, 用于厌氧, 或在其他环境中使用以极端分离具有独特特性的多肽。为某一特定目标确定的多肽和/或共识可作为活体材料, 或合成的细胞。合成的多肽可以进一步成熟的发展, 甚至更高的亲和力和更强健的蛋白质催化捕获 (PCC) 剂, 用于传感器, 或其他应用, 抗体通常会被用于捆绑或识别38,55。分子建模也可用于帮助确定肽与其目标的结合位置, 如最近在图 5C 1中所列的 abrax 结合肽和共识所执行的操作。总的来说, 筛选细菌显示库的肽亲和试剂是一个快速, 直接, 和强大的替代品生产抗体的识别和检测的蛋白质目标, 这半自动化筛选方法已取得了可靠的结果, 具有深远的应用。

筛选是一个证明的技术为亲和试剂发现, 与细菌显示图书馆一个方便来源为肽亲和性试剂1,2,3,4,5, 6,7,8,91011,1213,14 15,16,17,181920,2122,23 24。类似于噬菌体显示25,26和酵母显示27,28, 多肽在细胞表面上暴露, 并可绑定到生物和非生化目标, 与这些相互作用由肽骨架和氨基酸侧链的独特特性驱动的29。与噬菌体的显示相反, 细菌本身是自我复制的, 包含所有的遗传物质, 不需要洗脱的噬菌体和细胞的再感染。与酵母显示相比, 细菌显示更快 (约20分钟的30) 倍, 因为大肠杆菌的时间较快。所产生的肽亲和试剂可以在各种传感平台上产生非细胞, 16,17,26 , 并且在显示在单元格2时有可能用作生活材料。,31,32. 与用于识别特定目标的单克隆抗体相比 , 多肽更小、更灵活 , 而多肽显示库可以在 DNA 水平上很容易地纵 , 以避免特定的氨基酸 , 如半胱氨酸33.肽是越来越多的利用治疗34,35,36和其他应用程序, 抗体通常会被利用, 由于其易于发现, 可重复合成 (细胞) 在极端环境下的生产和优异的性能维护, 即使在高温或长期存储的情况下也不需要冷藏37,38, 也可以提供功能性试剂。克服冷链储存试剂的能力是国防部特别关心的, 例如, 它必须在极端环境下运作。因此, 热稳定性是一个可取的特点, 对亲和试剂识别选定的目标作为例子在这篇手稿。
最近, 筛选细菌显示库变得更加直接和可靠的使用半自动化磁激活细胞分类 (mac 或 MCS) 方法9,14,39。这些方法已经证明了生物目标使用几个类似的排序平台, 具有不同的优势, 包括一个商业可用的自动磁活化细胞分拣 (autoMACS 或 autoMCS) 仪器 (见表材料) 1,14,15和其他更专门的设备, 其中包含的样品在一次性墨盒7,9或芯片39, 一个有价值的规范使用生物安全等级 2 (BSL-2) 或更高的7的有机体或毒素。这些半自动的方法可以扩展到排序的多肽能够绑定到非生物材料, 如果材料是磁性或有能力被涂上磁珠, 并用于不同的生物体 (如厌氧菌), 如果排序和生长条件被改变。除了对细菌显示库进行分类外, 这里使用的商用 autoMCS 仪器还部分用于发现酵母表面显示的人类单链抗体片段的初步步骤, 然后进一步隔离使用荧光活化的细胞分类 (外地) 40。半自动化的主要优点是减少了分选时间和精力, 并且在洗涤步骤中更健壮和可重复地消除磁性珠子中的未绑定细胞, 导致误报率降低, 需要的分类轮数减少, 而复杂的下游分析9,14。在商业 autoMCS 仪器的情况下, 有多个预先加载的程序, 可能是最佳的不同的应用, 如负预排序 (损耗), 优先级纯度, 最小化最终样本量, 并增加或减少稀有单元隔离的灵敏度41。
这项工作的重点是演示筛选的方法, 使用商业上可用的 autoMCS 仪器的细菌显示库, 并阐述了思想的过程中应用在分析和尽量减少所产生的候选人池为了便于扩展到其他应用程序。此处使用的显示库 (参见资料表) 12,13包含大约 1091011独特的 15-滨海肽, 通过修改后的细菌外膜蛋白 OmpX 的版本显示在无约束性质的细胞表面, 这是预期的代表自由肽的解决方案, 如果综合生产。该蛋白支架还包含一个积极的控制 P2X 肽在 C-总站的监测表达通过结合到 YPet 莫娜。然而, 购买或新颖的图书馆具有适当的多样性和其他特性所需的具体应用, 应与此筛选程序类似, 虽然可能需要一些细微的变化。此筛选协议的示意图如图 1所示。此外, 该协议可以适应其他自动磁分拣平台和其他排序策略。手动磁选与台式磁铁已经成功地证明, 虽然更复杂和耗时的下游分析需要由于增加误报14, 但仍然提供了一个替代选项的autoMCS 步骤, 如果没有自动磁性细胞分拣设备可用。

<table>
	<tbody>
		<tr>
			<td>autoMACS Pro Separator</td>
			<td>Miltenyi Biotec</td>
			<td>130-092-545</td>
			<td>Automated cell separator for use with magnetic beads</td>
		</tr>
		<tr>
			<td>Luria Broth, Miller (LB)</td>
			<td>Fisher Scientific</td>
			<td>BP1426-500</td>
			<td>Growth medium</td>
		</tr>
		<tr>
			<td>Chloramphenicol</td>
			<td>Fisher BioReagents</td>
			<td>BP904-100</td>
			<td>Antibiotic</td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Fisher Scientific</td>
			<td>BP2641-500</td>
			<td>Thickening/geling agent</td>
		</tr>
		<tr>
			<td>eCPX 3.0 Bacterial Display Peptide Library</td>
			<td>Cytomx Therapeutics</td>
			<td>N/A; http://cytomx.com/contact/</td>
			<td>On-cell peptide display library. Provided by collaborators at USCB.</td>
		</tr>
		<tr>
			<td>L-arabinose</td>
			<td>Sigma</td>
			<td>A3256-500G</td>
			<td>Sugar, for induction of protein expression</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline pH 7.4 (PBS)</td>
			<td>Amresco</td>
			<td>0780-10L</td>
			<td>Buffer</td>
		</tr>
		<tr>
			<td>EZ-Link Sulfo-NHS-LC-Biotin, No-Weigh</td>
			<td>Thermo Scientific</td>
			<td>PI21327</td>
			<td>Adds biotin molecule to primary amines</td>
		</tr>
		<tr>
			<td>Pierce Biotin Quantitation Kit</td>
			<td>Thermo Scientific</td>
			<td>PI28005</td>
			<td>Ensures biotin molecule is covalently bound to protein</td>
		</tr>
		<tr>
			<td>Dynabeads MyOne Strepavidin T1 Beads</td>
			<td>Invitrogen</td>
			<td>65601</td>
			<td>Magnetic beads for capture of biotin-congugated proteins</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma</td>
			<td>A9647-100G</td>
			<td>Protein, used as blocking agent for non-specific binding</td>
		</tr>
		<tr>
			<td>D-glucose</td>
			<td>Sigma</td>
			<td>G8270-1KG</td>
			<td>Sugar, for growth and impeding inducible protein expression</td>
		</tr>
		<tr>
			<td>Ypet Mona</td>
			<td>UCSB and ARL</td>
			<td>N/A</td>
			<td>Positive control for monitoring expression of eCPX 3.0. Produced by authors and collaborators; see references 12 and 13.</td>
		</tr>
		<tr>
			<td>Dylight 488 NHS Ester</td>
			<td>Thermo Scientific</td>
			<td>46403</td>
			<td>For conjugating protein target to fluorophore</td>
		</tr>
		<tr>
			<td>Streptavidin, R-Phycoerythrin conjugate (SAPE)</td>
			<td>Thermo Scientific</td>
			<td>S866</td>
			<td>Negative control for monitoring non-specific binding to streptavidin</td>
		</tr>
		<tr>
			<td>BD FACSFlow</td>
			<td>Becton, Dickinson, and Company</td>
			<td>342003</td>
			<td>Buffer for running FACS instrument</td>
		</tr>
		<tr>
			<td>autoMACS Columns</td>
			<td>Miltenyi Biotech</td>
			<td>130-021-101</td>
			<td>For MACS Separation</td>
		</tr>
		<tr>
			<td>autoMACS Running Buffer &#8211; MACS Separation Buffer</td>
			<td>Miltenyi Biotech</td>
			<td>130-091-221</td>
			<td>For autoMACS instrument maintenance. Hazard: contains 0.09% azide.</td>
		</tr>
		<tr>
			<td>autoMACS Pro Washing Solution</td>
			<td>Miltenyi Biotech</td>
			<td>130-092-987</td>
			<td>For autoMACS instrument maintenance</td>
		</tr>
		<tr>
			<td>70% Ethanol</td>
			<td>VWR</td>
			<td>E505-4L</td>
			<td>Decontamination of autoMACS</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma</td>
			<td>G6279-1L</td>
			<td>For bacterial freezer stocks</td>
		</tr>
		<tr>
			<td>Chill 15 rack</td>
			<td>Miltenyi Biotec</td>
			<td>130-092-952</td>
			<td>For MACS Separation</td>
		</tr>
		<tr>
			<td>BD FACSCanto II</td>
			<td>Becton, Dickinson, and Company</td>
			<td>744810; http://www.bdbiosciences.com/us/instruments/research/cell-analyzers/bd-facscanto-ii/m/744810/overview</td>
			<td>For assessment of peptide binding to target</td>
		</tr>
		<tr>
			<td>BD FACSDiva Software</td>
			<td>Becton, Dickinson, and Company</td>
			<td>659523</td>
			<td>For assessment of peptide binding to target</td>
		</tr>
		<tr>
			<td>Dynabeads MPC-S magnetic particle concentrator</td>
			<td>Thermo Scientific</td>
			<td>A13346</td>
			<td>Bench top cell separator for use with magnetic beads</td>
		</tr>
		<tr>
			<td>Colony Sequencing</td>
			<td>Genewiz</td>
			<td>N/A; https://www.genewiz.com/</td>
			<td>DNA and Colony Sequencing Services</td>
		</tr>
		<tr>
			<td>Excel</td>
			<td>Microsoft</td>
			<td>323021400; https://www.microsoftstore.com/store/msusa/en_US/pdp/Excel-2016/productID.323021400</td>
			<td>For use with sequence analysis macro</td>
		</tr>
		<tr>
			<td>Anthrax Protective Antigen (PA)</td>
			<td>List Biological Laboratories, Inc.</td>
			<td>171</td>
			<td>Target protein used as example for representative results.</td>
		</tr>
		<tr>
			<td>Abrax</td>
			<td>ECBC and ARL</td>
			<td>N/A</td>
			<td>Target protein used as example for representative results. Produced by authors and collaborators; see references 1 and 15.</td>
		</tr>
		<tr>
			<td>Rivax</td>
			<td>ECBC and ARL</td>
			<td>N/A</td>
			<td>Target protein used as example for representative results. Produced by authors and collaborators; see references 1 and 15.</td>
		</tr>
	</tbody>
</table>

semi-automated biopanning of bacterial display libraries for peptide affinity reagent discovery and analysis of resulting isolates

筛选细菌显示库是一种成熟的技术, 用于多肽亲和试剂的发现, 既能识别生物目标, 也可用于非生化指标。肽亲和试剂可以用于类似的抗体的应用, 包括传感和治疗, 但更健壮, 能够在更极端的环境中执行。利用半自动化分选方法提高了多肽捕获剂对蛋白质目标的特异性, 改善了结合和洗涤步骤, 从而减少了假阳性粘合剂的发生。本文介绍了一种半自动的分拣方法, 它与一个具有无约束细菌显示分拣库的商用自动磁激活细胞分拣装置一起使用, 它表达了随机15多肽。稍加修改, 这些方法可扩展到其他自动化设备、其他分类库和其他有机体。这项工作的一个主要目标是提供一个全面的方法, 并阐述了在分析和尽量减少所产生的候选人库中应用的思维过程。这些技术包括使用荧光活化细胞分类 (外地) 对细胞结合进行分析, 在分类和比较个别候选者时评估亲和性和特异性, 并分析肽序列以确定趋势和共识序列的理解和潜在改善的亲和力和针对性的目标利益。

Watch this Scientific Journal Video about Semi-automated Biopanning of Bacterial Display Libraries for Peptide Affinity Reagent Discovery and Analysis of Resulting Isolates at JoVE.com

Semi-automated Biopanning of Bacterial Display Libraries for Peptide Affinity Reagent Discovery and Analysis of Resulting Isolates

筛选细菌显示库是一种被证实的技术, 用于发现肽亲和试剂, 一种强有力的替代抗体。本文的半自动排序方法简化了筛选, 减少了误报的发生。在这里, 我们说明的思想过程和技术应用于评价候选人和最小化下游分析。

细菌显示文库的半自动化筛选肽亲和试剂的发现与分离结果分析

Biopanning bacterial display libraries is a proven technique for peptide affinity reagent discovery for recognition of both biotic and abiotic targets. ...

semi-automated-biopanning-bacterial-display-libraries-for-peptide

Nanyang Technological University

Research

JoVE Journal

Biochemistry

11.3K 次观看.  US Army Research Laboratory. 筛选细菌显示库是一种被证实的技术, 用于发现肽亲和试剂, 一种强有力的替代抗体。本文的半自动排序方法简化了筛选, 减少了误报的发生。在这里, 我们说明的思想过程和技术应用于评价候选人和最小化下游分析。

视频: Semi-automated Biopanning of Bacterial Display Libraries for Peptide Affinity Reagent Discovery and Analysis of Resulting Isolates

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 ...

科研

生物化学

Bacterial Inner-membrane Display for Screening a Library of Antibody Fragments

Antibodies engineered for intracellular function must not only have affinity for their target antigen, but must also be soluble and correctly folded in the cytoplasm. Commonly used methods for the display and screening of recombinant antibody libraries do not incorporate intracellular protein folding quality control, and, thus, the antigen-binding capability and cytoplasmic folding and solubility of antibodies engineered using these methods often must be engineered separately. Here, we describe a protocol to screen a recombinant library of single-chain variable fragment (scFv) antibodies for antigen-binding and proper cytoplasmic folding simultaneously. The method harnesses the intrinsic intracellular folding quality control mechanism of the Escherichia coli twin-arginine translocation (Tat) pathway to display an scFv library on the E. coli inner membrane. The Tat pathway ensures that only soluble, well-folded proteins are transported out of the cytoplasm and displayed on the inner membrane, thereby eliminating poorly folded scFvs prior to interrogation for antigen-binding. Following removal of the outer membrane, the scFvs displayed on the inner membrane are panned against a target antigen immobilized on magnetic beads to isolate scFvs that bind to the target antigen. An enzyme-linked immunosorbent assay (ELISA)-based secondary screen is used to identify the most promising scFvs for additional characterization. Antigen-binding and cytoplasmic solubility can be improved with subsequent rounds of mutagenesis and screening to engineer antibodies with high affinity and high cytoplasmic solubility for intracellular applications.

We provide a method to simultaneously screen a library of antibody fragments for binding affinity and cytoplasmic solubility by using the Escherichia coli twin-arginine translocation pathway, which has an inherent quality control mechanism for intracellular protein folding, to display the antibody fragments on the inner membrane.

细菌内膜显示筛查抗体片段的图书馆

Antibodies engineered for intracellular function must not only have affinity for their target antigen, but must also be soluble and correctly folded in ...

Determination of High-affinity Antibody-antigen Binding Kinetics Using Four Biosensor Platforms

Label-free optical biosensors are powerful tools in drug discovery for the characterization of biomolecular interactions. In this study, we describe the use of four routinely used biosensor platforms in our laboratory to evaluate the binding affinity and kinetics of ten high-affinity monoclonal antibodies (mAbs) against human proprotein convertase subtilisin kexin type 9 (PCSK9). While both Biacore T100 and ProteOn XPR36 are derived from the well-established Surface Plasmon Resonance (SPR) technology, the former has four flow cells connected by serial flow configuration, whereas the latter presents 36 reaction spots in parallel through an improvised 6 x 6 crisscross microfluidic channel configuration. The IBIS MX96 also operates based on the SPR sensor technology, with an additional imaging feature that provides detection in spatial orientation. This detection technique coupled with the Continuous Flow Microspotter (CFM) expands the throughput significantly by enabling multiplex array printing and detection of 96 reaction sports simultaneously. In contrast, the Octet RED384 is based on the BioLayer Interferometry (BLI) optical principle, with fiber-optic probes acting as the biosensor to detect interference pattern changes upon binding interactions at the tip surface. Unlike the SPR-based platforms, the BLI system does not rely on continuous flow fluidics; instead, the sensor tips collect readings while they are immersed in analyte solutions of a 384-well microplate during orbital agitation.
Each of these biosensor platforms has its own advantages and disadvantages. To provide a direct comparison of these instruments&#39; ability to provide quality kinetic data, the described protocols illustrate experiments that use the same assay format and the same high-quality reagents to characterize antibody-antigen kinetics that fit the simple 1:1 molecular interaction model.

We describe here protocols for the measurement of antibody-antigen binding affinity and kinetics using four commonly used biosensor platforms.

高亲和力抗体 - 抗原的结合动力学的测定，使用四个生物传感器平台

Label-free optical biosensors are powerful tools in drug discovery for the characterization of biomolecular interactions. In this study, we describe the ...

Easy Manipulation of Architectures in Protein-based Hydrogels for Cell Culture Applications

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field of 3D templates is rising due to the potential resemblance of those materials to the natural extracellular matrix. Protein-based hydrogels are particularly promising because they can easily be functionalized and can achieve defined structures with adjustable physicochemical properties. However, the production of macroporous 3D templates for cell culture applications using natural materials is often limited by their weaker mechanical properties compared to those of synthetic materials. Here, different methods were evaluated to produce macroporous bovine serum albumin (BSA)-based hydrogel systems, with adjustable pore sizes in the range of 10 to 70 &#181;m in radius. Furthermore, a method to generate channels in this protein-based material that are several hundred microns long was established. The different methods to produce pores, as well as the influence of pore size on material properties such as swelling ratio, pH, temperature stability, and enzymatic degradation behavior, were analyzed. Pore sizes were investigated in the native, swollen state of the hydrogels using confocal laser scanning microscopy. The feasibility for cell culture applications was evaluated using a cell-adhesive RGD peptide modification of the protein system and two model cell lines: human breast cancer cells (A549) and adenocarcinomic human alveolar basal epithelial cells (MCF7).

Different methods to manipulate three-dimensional architecture in protein-based hydrogels are evaluated here with respect to material properties. The macroporous networks are functionalized with a cell-adhesive peptide, and their feasibility in cell culture is evaluated using two different model cell lines.

便于操作的体系结构基于蛋白质的凝胶中为单元格文化应用程序

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field ...

Analysis of Histone Antibody Specificity with Peptide Microarrays

Post-translational modifications (PTMs) on histone proteins are widely studied for their roles in regulating chromatin structure and gene expression. The mass production and distribution of antibodies specific to histone PTMs has greatly facilitated research on these marks. As histone PTM antibodies are key reagents for many chromatin biochemistry applications, rigorous analysis of antibody specificity is necessary for accurate data interpretation and continued progress in the field. This protocol describes an integrated pipeline for the design, fabrication and use of peptide microarrays for profiling the specificity of histone antibodies. The design and analysis aspects of this procedure are facilitated by ArrayNinja, an open-source and interactive software package we recently developed to streamline the customization of microarray print formats. This pipeline has been used to screen a large number of commercially available and widely used histone PTM antibodies, and data generated from these experiments are freely available through an online and expanding Histone Antibody Specificity Database. Beyond histones, the general methodology described herein can be applied broadly to the analysis of PTM-specific antibodies.

This manuscript describes methods for applying peptide microarray technology to specificity profiling of antibodies that recognize histones and their post-translational modifications.

用肽微阵列分析组蛋白抗体特异性

Post-translational modifications (PTMs) on histone proteins are widely studied for their roles in regulating chromatin structure and gene expression. The ...

Extracellular Protein Microarray Technology for High Throughput Detection of Low Affinity Receptor-Ligand Interactions

Secreted factors, membrane-tethered receptors, and their interacting partners are main regulators of cellular communication and initiation of signaling cascades during homeostasis and disease, and as such represent prime therapeutic targets. Despite their relevance, these interaction networks remain significantly underrepresented in current databases; therefore, most extracellular proteins have no documented binding partner. This discrepancy is primarily due to the challenges associated with the study of the extracellular proteins, including expression of functional proteins, and the weak, low affinity, protein interactions often established between cell surface receptors. The purpose of this method is to describe the printing of a library of extracellular proteins in a microarray format for screening of protein-protein interactions. To enable detection of weak interactions, a method based on multimerization of the query protein under study is described. Coupled to this microbead-based multimerization approach for increased multivalency, the protein microarray allows robust detection of transient protein-protein interactions in high throughput. This method offers a rapid and low sample consuming-approach for identification of new interactions applicable to any extracellular protein. Protein microarray printing and screening protocol are described. This technology will be useful for investigators seeking a robust method for discovery of protein interactions in the extracellular space.

Here, we present a protocol to screen extracellular protein microarrays for identification of novel receptor-ligand interactions in high throughput. We also describe a method to enhance detection of transient protein-protein interactions by using protein-microbead complexes.

低亲和力受体-配体相互作用的细胞外蛋白微阵列技术

Secreted factors, membrane-tethered receptors, and their interacting partners are main regulators of cellular communication and initiation of signaling ...

Real-Time, Semi-Automated Fluorescent Measurement of the Airway Surface Liquid pH of Primary Human Airway Epithelial Cells

In recent years, the importance of mucosal surface pH in the airways has been highlighted by its ability to regulate airway surface liquid (ASL) hydration, mucus viscosity and activity of antimicrobial peptides, key parameters involved in innate defense of the lungs. This is of primary relevance in the field of chronic respiratory diseases such as cystic fibrosis (CF) where these parameters are dysregulated. While different groups have studied ASL pH both in vivo and in vitro, their methods report a relatively wide range of ASL pH values and even contradictory findings regarding any pH differences between non-CF and CF cells. Furthermore, their protocols do not always provide enough details in order to ensure reproducibility, most are low throughput and require expensive equipment or specialized knowledge to implement, making them difficult to establish in most labs. Here we describe a semi-automated fluorescent plate reader assay that enables the real-time measurement of ASL pH under thin film conditions that more closely resemble the in vivo situation. This technique allows for stable measurements for many hours from multiple airway cultures simultaneously and, importantly, dynamic changes in ASL pH in response to agonists and inhibitors can be monitored. To achieve this, the ASL of fully differentiated primary human airway epithelial cells (hAECs) are stained overnight with a pH-sensitive dye in order to allow for the reabsorption of the excess fluid to ensure thin film conditions. After fluorescence is monitored in the presence or absence of agonists, pH calibration is performed in situ to correct for volume and dye concentration. The method described provides the required controls to make stable and reproducible ASL pH measurements, which ultimately could be used as a drug discovery platform for personalized medicine, as well as adapted to other epithelial tissues and experimental conditions, such as inflammatory and/or host-pathogen models.

We present a protocol to make dynamic measurements of the airway surface liquid pH under thin film conditions using a plate-reader.

一次人气道上皮细胞气道表面液体 pH 值的实时、半自动荧光测量

In recent years, the importance of mucosal surface pH in the airways has been highlighted by its ability to regulate airway surface liquid (ASL) ...

Native Cell Membrane Nanoparticles System for Membrane Protein-Protein Interaction Analysis

Protein-protein interactions in cell membrane systems play crucial roles in a wide range of biological processes- from cell-to-cell interactions to signal transduction; from sensing environmental signals to biological response; from metabolic regulation to developmental control. Accurate structural information of protein-protein interactions is crucial for understanding the molecular mechanisms of membrane protein complexes and for the design of highly specific molecules to modulate these proteins. Many in vivo and in vitro approaches have been developed for the detection and analysis of protein-protein interactions. Among them the structural biology approach is unique in that it can provide direct structural information of protein-protein interactions at the atomic level. However, current membrane protein structural biology is still largely limited to detergent-based methods. The major drawback of detergent-based methods is that they often dissociate or denature membrane protein complexes once their native lipid bilayer environment is removed by detergent molecules. We have been developing a&#160;native cell membrane nanoparticle system for membrane protein structural biology. Here, we demonstrate the use of this system in the analysis of protein-protein interactions on the cell membrane with a case study of the oligomeric state of AcrB.

Presented here is a protocol for the determination of oligomeric state of membrane proteins that utilizes a native cell membrane nanoparticle system in conjunction with electron microscopy.

用于膜蛋白-蛋白质相互作用分析的原生细胞膜纳米粒子系统

Protein-protein interactions in cell membrane systems play crucial roles in a wide range of biological processes- from cell-to-cell interactions to signal ...

Using Phage Display to Develop Ubiquitin Variant Modulators for E3 Ligases

Ubiquitin is a small 8.6 kDa protein that is a core component of the ubiquitin-proteasome system. Consequently, it can bind to a diverse array of proteins with high specificity but low affinity. Through phage display, ubiquitin variants (UbVs) can be engineered such that they exhibit improved affinity over wildtype ubiquitin and maintain binding specificity to target proteins. Phage display utilizes a phagemid library, whereby the pIII coat protein of a filamentous M13 bacteriophage (chosen because it is displayed externally on the phage surface) is fused with UbVs. Specific residues of human wildtype ubiquitin are soft and randomized (i.e., there is a bias towards to native wildtype sequence) to generate UbVs so that deleterious changes in protein conformation are avoided while introducing the diversity necessary for promoting novel interactions with the target protein. During the phage display process, these UbVs are expressed and displayed on phage coat proteins and panned against a protein of interest. UbVs that exhibit favorable binding interactions with the target protein are retained, whereas poor binders are washed away and removed from the library pool. The retained UbVs, which are attached to the phage particle containing the UbV's corresponding phagemid, are eluted, amplified, and concentrated so that they can be panned against the same target protein in another round of phage display. Typically, up to five rounds of phage display are performed, during which a strong selection pressure is imposed against UbVs that bind weakly and/or promiscuously so that those with higher affinities are concentrated and enriched. Ultimately, UbVs that demonstrate higher specificity and/or affinity for the target protein than their wildtype counterparts are isolated and can be characterized through further experiments.

Ubiquitination is a critical protein post-translational modification, dysregulation of which has been implicated in numerous human diseases. This protocol details how phage display can be utilized to isolate novel ubiquitin variants that can bind and modulate the activity of E3 ligases that control the specificity, efficiency, and patterns of ubiquitination.

使用噬菌体显示开发用于E3连接酶的泛素变体调节剂

Ubiquitin is a small 8.6 kDa protein that is a core component of the ubiquitin-proteasome system. Consequently, it can bind to a diverse array of proteins ...

Bacterial Expression and Purification of Human Matrix Metalloproteinase-3 using Affinity Chromatography

Matrix metalloproteinases (MMPs) belong to the family of metzincin proteases with central roles in extracellular matrix (ECM) degradation and remodeling, as well as interactions with several growth factors and cytokines. Overexpression of specific MMPs is responsible in several diseases such as cancer, neurodegenerative diseases, and cardiovascular disease. MMPs have been the center of attention recently as targets to develop therapeutics that can treat diseases correlated to MMP overexpression.
To study the MMP mechanism in solution, more facile and robust recombinant protein expression and purification methods are needed for the production of active, soluble MMPs. However, the catalytic domain of most MMPs cannot be expressed in Escherichia coli (E. coli) in soluble form due to lack of posttranslational machinery, whereas mammalian expression systems are usually costly and have lower yields. MMP inclusion bodies must undergo the tedious and laborious process of extensive purification and refolding, significantly reducing the yield of MMPs in native conformation. This paper presents a protocol using Rosetta2(DE3)pLysS (hereafter referred to as R2DP) cells to produce matrix metalloproteinase-3 catalytic domain (MMP-3cd), which contains an N-terminal His-tag followed by pro-domain (Hisx6-pro-MMP-3cd) for use in affinity purification. R2DP cells enhance the expression of eukaryotic proteins through a chloramphenicol-resistant plasmid containing codons normally rare in bacterial expression systems. Compared to the traditional cell line of choice for recombinant protein expression, BL21(DE3), purification using this new strain improved the yield of purified Hisx6-pro-MMP-3cd. Upon activation and desalting, the pro domain is cleaved along with the N-terminal His-tag, providing active MMP-3cd for immediate use in countless in vitro applications. This method does not require expensive equipment or complex fusion proteins and describes rapid production of recombinant human MMPs in bacteria.

His-tag purification, dialysis, and activation are employed to increase yields of soluble, active matrix metalloproteinase-3 catalytic domain protein expression in bacteria. Protein fractions are analyzed via SDS-PAGE gels.

亲和色谱法表达和纯化人基质金属蛋白酶-3

Matrix metalloproteinases (MMPs) belong to the family of metzincin proteases with central roles in extracellular matrix (ECM) degradation and remodeling, ...