蛋白质直接送货到哺乳动物细胞利用细胞渗透半胱氨酸<sub> 2</sub> -His<sub> 2</sub>锌指域
Summary
锌指结构域是本质细胞渗透性和能够介导蛋白输送到广泛的哺乳动物细胞类型的。这里，一个详细的一步一步的协议用于实现锌指技术为细胞内蛋白输送呈现。
Abstract
Due to their modularity and ability to be reprogrammed to recognize a wide range of DNA sequences, Cys2-His2 zinc-finger DNA-binding domains have emerged as useful tools for targeted genome engineering. Like many other DNA-binding proteins, zinc-fingers also possess the innate ability to cross cell membranes. We recently demonstrated that this intrinsic cell-permeability could be leveraged for intracellular protein delivery. Genetic fusion of zinc-finger motifs leads to efficient transport of protein and enzyme cargo into a broad range of mammalian cell types. Unlike other protein transduction technologies, delivery via zinc-finger domains does not inhibit enzyme activity and leads to high levels of cytosolic delivery. Here a detailed step-by-step protocol is presented for the implementation of zinc-finger technology for protein delivery into mammalian cells. Key steps for achieving high levels of intracellular zinc-finger-mediated delivery are highlighted and strategies for maximizing the performance of this system are discussed.
Introduction
高效，多功能的蛋白质交付策略是许多基础研究和治疗应用的关键。直接输送纯化的蛋白质导入细胞代表一个的最安全和最容易的方法来实现这一点。1,2-不同于依靠从核酸的基因表达的策略，3-5蛋白输送带来插入突变的危险，是独立的细胞转录/翻译机制，并允许立竿见影的效果。然而，由于缺乏简单，可推广的方法赋予细胞渗透活性的蛋白质上经常混淆其直接进入细胞。目前的方法用于促进细胞内蛋白质递送的基于使用的天然存在的6-8或设计的细胞穿透肽，9-12增压转导结构域，13,14纳米颗粒15和脂质体，16病毒样颗粒17,18 </SUP>和聚合物微球材料。19不幸的是，许多这些方法是由低细胞摄取速率，20,21稳定性差，22无意细胞类型特异性，低23内体逃逸属性24和 毒性。25除了受阻，许多蛋白质转导技术减少递送蛋白质的生物活性。14
我们实验室以前表明，锌指核酸酶（ZFN）的蛋白质-嵌合限制性内切酶选自由可编程的Cys 2 -His 2锌指DNA结合蛋白和的FokⅠ限制性内切酶26-28的切割结构域的-是固有小区可渗透的。29这种令人惊奇的细胞穿透活性被证明是定制设计的锌指结构域，即已经成为一个强大的工具用于靶向基因组中的连接DNA结合平台的固有性质工程设计，30-32，被认为是六对蛋白质表面带正电荷的残基的构象的结果。实际上，一些DNA结合蛋白，包括c-Jun的和N-DEK已显示具有穿过细胞膜的固有能力。33最近，我们的实验室扩展了这些结果，并表明，锌的细胞穿透活性手指（ZIF）域，可以利用细胞内蛋白质传递。遗传融合任一个或两个手指ZIF域特异性蛋白货物导致摄取效率超出许多常规细胞穿透肽递送系统34最值得注意的是，ZIF介导的递送没有妥协的稠酶促货物的活性和促进高水平的胞浆交付。总的来说，这些研究结果表明该ZIF域的电位用于促进高效和轻便递送的蛋白质，和宏观的潜在更加多样类型分子进入细胞。
在这里，一个详细的一步一步的协议，就如何落实ZIF技术蛋白交付在哺乳动物细胞中被提出。我们以前建造一套一，二，三，四，五，六指缺乏结合DNA，由于每个α螺旋DNA结合残基的替代能力ZIF域，但能够提供蛋白质进入细胞34（ 图1）。翡翠绿色荧光蛋白（EmGFP）的制备和转导入用两个手指ZIF域HeLa细胞进行说明。这个协议是可扩展的，以几乎能够在大肠杆菌和可溶性表达几乎任何哺乳动物细胞类型的任何蛋白质。提供预期结果，并且还讨论了用于最大化该系统的性能的策略。
Protocol
Representative Results
Discussion
这里，一步一步协议用于使用细胞渗透性锌指（ZIF）结构域蛋白输送呈现。该ZIF域不降低融合酶的货物34活动;允许生产和收益率几乎相同的那些与未修饰蛋白观察到的蛋白质的纯化;和可运输的蛋白质和酶，用于广泛范围的细胞类型，效率超过传统的细胞穿透肽或蛋白质转导结构域的系统。总之，这些发现表明ZIF域的广泛的潜在介导的直接蛋白输送到细胞内的广泛的应用范围。
<p class="j…Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作是由美国国立卫生研究院（DP1CA174426卡洛斯F.巴尔巴斯）和上海科技大学，上海，中国（以JL）的支持。使用PyMOL的生成分子图形。
Materials
| XmaI | New England Biolabs | R0180L | |
| SacI | New England Biolabs | R0156L | |
| Expand High Fidelity PCR system | Roche | 11759078001 | |
| dNTPs | New England Biolabs | N0446S | |
| 4-20% Tris-Glycine Mini protein gels, 1.5 mm, 10 wells | Life Technologies | EC6028BOX | |
| 2x Laemmli Sample Buffer | BioRad | 161-0737 | |
| T4 DNA Ligase | Life Technologies | 15224-017 | |
| BL21 (DE3) Competent E. coli | New England Biolabs | C2527I | |
| IPTG | Thermo Scientific | R0391 | |
| Zinc Chloride | Sigma-Aldrich | 208086-5G | |
| Kanamycin Sulfate | Fisher Scientific | BP906-5 | |
| Glucose | Sigma-Aldrich | G8270-100G | |
| Tris Base | Fisher Scientific | BP152-25 | |
| Sodium Chloride | Sigma-Aldrich | S9888-25G | |
| DTT | Fisher Scientific | PR-V3151 | |
| PMSF | Thermo Scientific | 36978 | |
| Ni-NTA Agarose Resin | QIAGEN | 30210 | |
| Glycerol | Sigma-Aldrich | G5516-500ML | |
| Imidazole | Sigma-Aldrich | I5513-25G | |
| Amicon Ultra-15 Centrifugal Filter Units | EMO Millipore | UFC900324 | |
| DMEM | Life Technologies | 11966-025 | |
| Fetal Bovine Serum | Life Technologies | 10437-028 | |
| Antibiotic-Antimycotic | Life Technologies | 15240-062 | |
| 24-Well Flat Bottom Plate | Sigma-Aldrich | CLS3527-100EA | |
| Poly-Lysine | Sigma-Aldrich | P7280 | |
| DPBS, No Calcium, No Magnesium | Life Technologies | 21600010 | |
| Heparan Sulfate | Sigma-Aldrich | H4777 | |
| Trypsin | Life Technologies | 25300054 | |
| Hela cells | ATCC | CCL-2 | |
| Nano Drop ND-1000 spectrophotometer | Thermo Fisher Scientific | N/A | |
| QIAquick PCR Purification Kit | QIAGEN | 28104 | |
| QIAquick Gel Extraction Kit | QIAGEN | 28704 |
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