使用噬菌体显示开发用于E3连接酶的泛素变体调节剂
Summary
泛素化是翻译后修饰的关键蛋白质，其失调与许多人类疾病有关。该协议详细介绍了如何利用噬菌体显示来分离新的泛素变体，这些变体可以结合和调节控制泛素特异性，效率和模式的E3连接酶的活性。
Abstract
泛素是一种8.6 kDa的小蛋白，是泛素蛋白酶体系统的核心成分。因此，它可以与具有高特异性但低亲和力的各种蛋白质结合。通过噬菌体展示，可以设计泛素变体（UbV），使它们表现出比野生型泛素更好的亲和力，并保持与靶蛋白的结合特异性。噬菌体展示利用噬菌体文库，其中丝状M13噬菌体的pIII包衣蛋白（选择它是因为它显示在噬菌体表面上）与UbV融合。人类野生型泛素的特定残基是软的和随机的（即，偏向于天然野生型序列）以产生UbVs，从而避免蛋白质构象的有害变化，同时引入促进与靶蛋白的新相互作用所需的多样性。在噬菌体展示过程中，这些UbV在噬菌体涂层蛋白上表达和显示，并针对感兴趣的蛋白质进行平移。与靶蛋白表现出有利结合相互作用的UbV被保留，而较差的结合剂被冲走并从文库池中除去。保留的UbV附着在含有UbV相应噬菌体颗粒上的UbV被洗脱，扩增和浓缩，以便它们可以在另一轮噬菌体展示中与相同的靶蛋白平移。通常，最多进行五轮噬菌体展示，在此期间，对弱结合和/或混杂结合的UbV施加强大的选择压力，以便集中和富集具有较高亲和力的UbV。最终，分离出比野生型对应物对靶蛋白具有更高特异性和/或亲和力的UbV，并且可以通过进一步的实验进行表征。
Introduction
了解蛋白质 – 蛋白质相互作用的分子细节对于描绘生物过程的信号转导机制至关重要，特别是那些导致临床重要疾病的机制。近年来，噬菌体展示已被用作分离蛋白质/肽的实用且易于使用的方法，其与所需目标蛋白的结合得到了很大的改善1，2，3，4，而靶蛋白相互作用又可用作蛋白 – 蛋白相互作用的细胞内探针。
泛素化是酶促活性（E1激活酶→E2偶联酶→E3连接酶）的级联反应，其共价偶联泛素（Ub）到蛋白质底物以靶向它们进行降解或介导细胞信号传导变化。此外，去泛素酶催化从蛋白质中去除泛素。因此，在细胞中，有成千上万的Ub依赖性蛋白质 – 蛋白质相互作用，其中绝大多数识别具有低亲和力但高特异性的共同表面，以允许通过大而多样化的表面进行弱相互作用。
Ernst等人将突变引入Ub的已知结合区域，以查看它们是否可以增强对目标蛋白质的结合亲和力，同时仍保持高选择性5。开发了一个超过100亿（7.5 x 1010）Ub变体（UbVs）的组合库，这些变体在Ub表面的位置发生了突变，介导了已知的Ub蛋白相互作用。该文库由表达融合到多样化UbV的M13噬菌体pIII涂层蛋白的噬菌体组成。因此，单个UbV可以在表达时通过包衣蛋白显示在噬菌体表面上。在选择过程中，显示与目标蛋白具有显着结合相互作用的UbV的噬菌体将在随后的噬菌体显示轮中被保留和富集，而显示与靶蛋白结合不良的UbV的噬菌体将被冲走并从噬菌体池中去除。保留的噬菌体颗粒含有与其显示的UbV相对应的噬菌体粒，允许它们在分离后进行测序和进一步表征。
利用这种蛋白质工程策略，开发了用于人类去泛素酶5和病毒蛋白酶的UbV抑制剂6。重要的是，我们通过劫持E2结合位点并激活占据HECT结构域上Ub结合外源的UbVs，为人类HECT家族E3连接酶生成了抑制性UbV7。我们还可以通过靶向E2结合位点来抑制单体环家族E3s，并诱导UbV二聚化以激活同源二聚体RING E3s8。对于多亚基RING E3s，UbVs可以通过靶向RING亚基（例如，对于APC / C复合物9）或破坏复合物形成（例如，对于SCF E3s10）来实现抑制。总的来说，可以利用UbV来系统地询问Ub蛋白酶体系统（UPS）中的蛋白质 – 蛋白质相互作用，以便我们可以更好地破译UPS酶的生化机制，并识别和验证治疗干预的功能位点。
以下方案描述了如何利用先前生成的Phage显示的UbV文库来靶向感兴趣的蛋白质，以及如何通过连续几轮噬菌体显示来富集与目标蛋白质相互作用的UbV结合剂。
Protocol
Representative Results
Discussion
如步骤2.1（蛋白质制备）中所述，可以使用多种方法来评估蛋白质浓度，并且每种方法都将基于用于噬菌体显示的特定靶蛋白具有独特的优缺点。以前已经提供了流行方法的详细描述和协议的来源11。
使用上一轮噬菌体展示保留的噬菌体作为后续一轮的输入，通过逐渐去除结合微弱、瞬时或偶然结合的粘结剂来丰富良好的粘结剂。到第四轮和第五轮，理想情…
Declarações
The authors have nothing to disclose.
Acknowledgements
泛素变体技术是在Sachdev Sidhu博士（多伦多大学）的实验室中设计的。WZ目前是人类与微生物组计划的CIFAR Azrieli全球学者。这项研究由授予WZ的NSERC发现补助金（RGPIN-2019-05721）资助。
Materials
| Axygen Mini Tube System (0.65 mL, sterile, 96/Rack, 10 Racks/pack) | Fisher Scientific | 14-222-198 | Culturing phage outputs after phage display. |
| BD Difco Dehydrated Culture Media: LB Broth, Miller (Luria-Bertani) | Fisher Scientific | DF0446-17-3 | Preparing plates for titering. |
| Bovine Serum Albumin (BSA), Fraction V | BioShop Canada | ALB001 | Buffer component. |
| Carbenicillin disodium salt 89.0-100.5% anhydrous | Millipore-Sigma | C1389-5G | Culturing phagemid-infected cells. |
| Compact Digital Microplate Shaker | Fisher Scientific | 11-676-337 | Shaking plates during incubation with the phage library. |
| Corning Microplate Aluminum Sealing Tape | Fisher Scientific | 07-200-684 | Sealing phage glycerol stocks. |
| Dehydrated Agar | Fisher Scientific | DF0140-01-0 | Preparing plates for titering. |
| DS-11 Spectrophotometer/Fluorometer | DeNovix | DS-11 FX+ | Protein concentration measurement. |
| Greiner Bio-One CellStar 96-Well, Non-Treated, U-Shaped-Bottom Microplate | Fisher Scientific | 7000133 | Storing phage glycerol stocks. |
| Hydrochloric Acid | Fisher Scientific | A144-500 | Phage elution. |
| Invitrogen One Shot OmniMAX 2 T1R Chemically Competent E. coli | Fisher Scientific | C854003 | Bacterial strain for phage infection. |
| Kanamycin Sulfate | Fisher Scientific | AAJ1792406 | Culturing M13K07 helper phage-infected cells. |
| M13KO7 Helper Phage | New England Biolabs | N0315S | Permit phagemid packing and secretion. |
| MaxQ 4000 Benchtop Orbital Shaker | Fisher Scientific | 11-676-076 | Bacterial cell culture. |
| Nunc MaxiSorp 96 well microplate, flat bottom | Life Technologies | 44-2404-21 | Immobilizing proteins. |
| Phosphate Buffered Saline (PBS) 10X Solution | Fisher Scientific | BP3994 | Buffer component/phage resuspension medium. |
| Polyester Films for ELISA and Incubation | VWR | 60941-120 | Covering the microplates during incubation. |
| Polyethylene Glycol 8000 (PEG) | Fisher Scientific | BP233-1 | Phage precipitation. |
| Sodium chloride | Millipore-Sigma | S3014 | Phage precipitation. |
| Sterile Plastic Culture Tubes: Translucent Polypropylene | Fisher Scientific | 14-956-1D | Culturing phage inputs. |
| Tetracycline Hydrochloride | Fisher Scientific | BP912-100 | Culturing E. coli OmniMax cells. |
| Tris Base | Fisher Scientific | BP1525 | Neutralizing eluted phage solution. |
| Tryptone Powder | Fisher Scientific | BP1421-2 | Cell growth media component. |
| Tween 20 | Fisher Scientific | BP337500 | Buffer component. |
| Yeast Extract | Fisher Scientific | BP1422-2 | Cell growth media component. |
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