高通量基因定位在<em>布氏锥虫</em
Summary
Addition of a tag to a protein is a powerful way of gaining insight into its function. Here, we describe a protocol to endogenously tag hundreds of Trypanosoma brucei proteins in parallel such that genome scale tagging is achievable.
Abstract
Improvements in mass spectrometry, sequencing and bioinformatics have generated large datasets of potentially interesting genes. Tagging these proteins can give insights into their function by determining their localization within the cell and enabling interaction partner identification. We recently published a fast and scalable method to generate Trypanosoma brucei cell lines that express a tagged protein from the endogenous locus. The method was based on a plasmid we generated that, when coupled with long primer PCR, can be used to modify a gene to encode a protein tagged at either terminus. This allows the tagging of dozens of trypanosome proteins in parallel, facilitating the large-scale validation of candidate genes of interest. This system can be used to tag proteins for localization (using a fluorescent protein, epitope tag or electron microscopy tag) or biochemistry (using tags for purification, such as the TAP (tandem affinity purification) tag). Here, we describe a protocol to perform the long primer PCR and the electroporation in 96-well plates, with the recovery and selection of transgenic trypanosomes occurring in 24-well plates. With this workflow, hundreds of proteins can be tagged in parallel; this is an order of magnitude improvement to our previous protocol and genome scale tagging is now possible.
Introduction
布氏锥虫是原生动物寄生虫引起牛非洲人类trypanosomasis和那加那T。布氏是蛋白质功能的分析的理想生物体由于高品质的基因组，众多蛋白质组学和转录数据集和发达分子工具1-3的组合。蛋白质组学和测序的进步导致了突出潜在的有趣的基因4-6大型数据集;然而，许多基因都与他们现有的数据库相关联的最少信息。因此，有需要一种高通量方法，以帮助蛋白质功能表征。
一个标签蛋白的表达能给见解的多样性成蛋白质的功能。例如，标记有荧光蛋白或表位的蛋白质可以通过荧光显微镜，其中给出了关于其中PROT信息进行本地化EIN可能会发挥其生物效应。另外，贴上了TAP 7的蛋白质，HaloTag 8或他的标签可以用于生化分析及其合作伙伴的互动鉴定纯化。
我们最近开发出一种强大的标注方法为T.布氏 9。这在过去很长的引物PCR生成转染DNA，并允许数十种蛋白质并行的标记 – 一个重大改进现有的协议。现在，我们已经通过了一个数量级提高该协议在procyclic形式的可扩展性。这里，我们提出我们的方法，其中，我们执行PCR和转染到96-孔板，以恢复和选择在24孔板中发生。数百蛋白质现在可以在并行进行标记该方法提供用于标记整个锥虫基因组中的具有成本效益的，可行的方法。
Protocol
Representative Results
Discussion
在蛋白质组学和转录方法在过去的5 – 10年的灵敏度显着改善提供了数以千计的基因及其产物的有价值的数据。然而，这些工具来解决这些蛋白质的功能没有跟上。
标记的蛋白质有利于无数次的实验，以确定其功能。例如，蛋白质可以在各种不同的颜色，以促进定位和共定位研究来融合于荧光蛋白。标签电子显微镜，如APEX2或miniSOG 11,12开发，使标签蛋白的超微结构定位。标签为生物化学，例如在TAP标记，并ProtC-TEV-PROTA（PTP）标记7,13允许与蛋白质的结合伙伴的身份或体外生物化学测定相关联配合物的纯化。
那些对protoc的成功至关重要的具体步骤醇有：二甲基亚砜掺入PCR主混合物1之前，在加入主混合物2中，在主混合物2中使用的商业聚合酶和cytomix电穿孔缓冲液的修饰的冻结PCR主要混合液1。在我们的经验，这是必要的，以便达到阳性孔的相似的比例使用细胞的双数为C末端标记的转染，作为N末端标记的转染。因此，如所描述，应执行的所有步骤。
这种技术只可能转染虫procyclic形式锥虫时获得成功。血液形式具有较低的转染效率14，而且他们很可能是由于这是无关的选择药物的密度依赖毒性选择过程中死亡。因此，我们以前的协议代表了血液形式锥虫9标记当前最先进的技术。它也可能是反式染效率会变化取决于特定的锥虫分离物。其他菌株可能需要额外的优化 – 这个协议使用927 SMOX procyclic形式进行了优化。可能增加成功的概率的措施包括：增加PCR扩增子的量，增加包括在转染细胞的数目。
我们提出了数百蛋白质可以并行标记的方法。这将有利于本地化和互动的大规模研究，补充现有大型数据集，并提供宝贵的信息社会。底漆模板可根据要求和质粒模板列表可以从：http://www.sdeanresearch.com/
Disclosures
The authors have nothing to disclose.
Acknowledgements
SD was supported by a Sir Henry Wellcome Fellowship [092201/Z/10/Z]. This work was funded by Wellcome Trust grants [WT066839MA][104627/Z/14/Z][108445/Z/15/Z] to Professor Keith Gull. We would like to thank Professor Keith Gull for helpful conversations and insights.
Materials
| Oligonucleotide | Life technologies | na | desalt only (do not use additional purification) |
| DMSO | Roche | Included with the Expand HiFi pack | Must be PCR grade |
| dNTP | any reputable | ||
| Expand HiFi polymerase | Roche | 11759078001 | |
| BTX ECM830 | Harvard Apparatus | Electroporator | |
| HT-200 plate handler | Harvard Apparatus | HT-200 | Handles the 96-well eletroplates |
| MOS 96 ELECTROPLATE 4mm | Harvard Apparatus | 45-0452 | Electroplate for the 96-well electroporation |
| Blasticidin S Hydrochloride | Melford | 3513-03-9 | |
| Hygromycin b Gold | Invivogen | ant-hg-1 | |
| Phleomycin | Melford | P0187 | |
| 225cm TC Flask, canted neck, phenolic cap | Appletonwoods | BC006 | |
| 24 well culture plates | Appletonwoods | BC017 | |
| Eppendorf® PCR Cooler, iceless cold storage system for 96 well plates and PCR tubes | Sigma Aldrich | Z606634-1EA | |
| 96-well Multiply® PCR plate with lateral skirt | Sarstedt | 72.1979.203 |
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