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。

1.克隆<ol><li>获取已亚克隆入PET-28的表达载体系统和可应要求提供（PET-2F-ZIF）丙氨酸取代的两个手指ZIF域。34 </li><li> PCR扩增EmGFP从质粒翡翠-的pBAD与引物的5&#39;XmaI-EmGFP（5&#39;-GGAAATTG CCCGGG ATGGTGAGCAAGGGCGAGGAGCTGTTCAC-3&#39;; XMA我粗体位点）和3&#39;SacI位-EmGFP（5&#39;-CGGATCT GAGCTC TTACTTGTACAGCTCGTCCATGCCGAG-3&#39; ;大胆囊我的网站）。 <ol><li>使用5纳克模板DNA，10微升10X聚合酶缓冲液，1单位（U）Taq DNA聚合酶，0.2mM的每种dNTP和0.2中与剩余的体积为100微升的溶液的每种引物μM由蒸馏/去离子水。使用的循环条件：95℃下5分钟;的95℃的30个循环持续30秒，55℃30秒和72℃进行1分钟; 72℃下进行10分钟。通过凝胶extracti纯化PCR产物上，并确定使用分光光度计测量绝对260×50微克/ ml的DNA浓度。 </li></ol></li><li>消化的pET-2F-ZIF并用限制性编码EmGFP插入酶XMA我和 SacI我在推荐的缓冲液3小时，在37℃用酶10单位每1微克的DNA。可视化的琼脂糖凝胶电泳使用荧光嵌入染料，如溴化乙锭的DNA。 </li><li>净化用凝胶提取试剂盒将消化的DNA，并确定由分光光度计测量绝对260×50微克/ ml的DNA浓度。 </li><li>结扎纯化EmGFP编码DNA成50-100纳克的pET-2F-ZIF使用T4 DNA连接酶的1单位在室温至少1小时。对于最好的结果，执行使用6的连接反应：1插入到载体的摩尔比。 </li><li>解冻50微升冰的任何化学感受XL-1蓝大肠杆菌细胞和10-20纳克连接PET-2F-ZIF-EmGFP的轻轻混匀。 </li><li>保持在冰上30分钟。热休克在42℃为90秒，该混合物回收细胞在2ml超级最优肉汤与分解代谢物阻抑（SOC），1小时，在37℃振荡。 </li><li>传播100微升恢复文化上的LB培养基（LB）琼脂平板上用50微克/毫升卡那霉素和孵化O / N在37℃。 </li><li>翌日，接种6毫升超级肉汤（SB）或含50微克/毫升卡那霉素与来自LB琼脂平板上并培养O / N一个菌落在37℃的LB培养物。 注意：在使用菌落PCR引物5&#39;XmaI-EmGFP和3&#39;SacI位-EmGFP可用于标识要Miniprep试剂之前阳性克隆。 </li><li>纯化的pET-2F-ZIF-EmGFP通过小量制备和使用T7启动子的DNA测序确认质粒（5&#39;-TAATACGACTCACTATAGGG-3&#39;）。 </li></ol> 2.表达和纯化<ol><li>解冻50微升化学感受BL21 E的大肠杆菌冰细胞和100纳克PL的PET-2F-ZIF-EmGFP轻轻混匀asmid。变换为在步骤1.7-1.8中描述。 </li><li>翌日，接种含有50微克/毫升卡那霉素具有单个菌落10毫升LB培养基和生长O / N在37℃振荡。 </li><li>第二天，稀释10毫升的O / N培养的成1升LB培养基中补充有50微克/ ml卡那霉素，0.2％葡萄糖和100微米的ZnCl 2。生长的培养，在37℃振荡的光学密度在0.8 600纳米（OD 600）和诱导蛋白表达有2mM异丙基β-D-1-硫代半乳糖苷（IPTG）。 6小时后的生长在37℃，收获细胞，离心，在5000×g离心10分钟，在4℃。 注意：感应条件是高度可变的并且依赖于蛋白质的稳定性被表达。监测OD 600每30分钟，直到OD达到600的0.8。 </li><li>重悬细胞沉淀在5ml裂解缓冲液（50mM的Tris-HCl，500mM的氯化钠，100μM的ZnCl 2，1米M二硫苏糖醇（DTT），1mM的MgCl 2的，1mM苯甲基磺酰氟（PMSF）和10mM咪唑，pH 8.0）中。裂解冰上的细胞超声具有以下设置：50％的动力输出，5秒开/ 10秒关闭间隔4分钟的处理时间。避免解决方案过热。 </li><li>离心细胞裂解物在25000×g离心30分钟，在4℃，将上清液转移到一个新的收集管。对于最好的结果，执行在4℃以下所有步骤。 </li><li>运行通过一列预填充用1ml平衡的Ni-NTA浆液的上清液。用20ml洗涤缓冲液（50mM的Tris-HCl，500mM的氯化钠，100μM的ZnCl 2，1mM的DTT，1mM的MgCl 2的和的30mM咪唑，pH 8.0）中洗涤树脂。 </li><li>洗脱用5ml洗脱缓冲液（50mM的Tris-HCl，500mM的氯化钠，100μM的ZnCl 2，1mM的DTT，1mM的MgCl 2的，和300 mM咪唑，pH8.0）中的蛋白质。 </li><li>缓冲液交换用缓冲存储器的洗脱的蛋白（50毫的Tris-HCl，500mM的氯化钠，100μM的ZnCl 2，1mM的DTT，1mM的MgCl 2的10％的甘油，pH 8.0）中，并使用根据制造商的说明书通过离心分离旋集中器集中的蛋白以至少40微米。 </li><li>用BCA或Bradford试验测定蛋白质浓度。拌2微升纯化的蛋白与2微升2×SDS-PAGE上样染料，煮沸，在95℃下进行10分钟，然后在4％解决-20％Tris-甘氨酸SDS-PAGE来评估蛋白质纯度（ 图2）。 </li></ol> 3.蛋白质存储<ol><li>分装浓缩的蛋白质，闪存冷冻在液氮中，并储存在-80℃。对于EmGFP融合蛋白，用铝箔覆盖的管中，以保护该蛋白质从光。 </li><li>避免反复冷冻和蛋白质溶液的解冻。注意：ZIF-融合蛋白是在这些条件下稳定至少1个月。不当或&gt;4个月存储可能会导致蛋白质沉淀或EmGFP的漂白。 </li></ol> 4.蛋白转导<ol><li>维持HeLa细胞中含有10％的Dulbecco氏改良的Eagle氏培养基（DMEM）（体积/体积）胎牛血清（FBS）和1％的抗生素-抗真菌剂在完全湿润气氛，用5％的CO 2在37℃。 注：细胞传代不推荐用于蛋白转导的30倍以上。 </li><li>预涂24孔板用500μl的多聚赖氨酸的50微克/毫升30至60分钟，在25℃下。种子细胞到24孔板，每孔2×10 5个细胞的密度。在接种后24小时，或一旦细胞是在80％-90％汇合，从各取出介质井和洗涤用500μl的预热无血清DMEM（SFM）。 </li><li>向每个孔中，加入含有2微米的ZIF-EmGFP蛋白和100μM的氯化锌加入250μlSFM的。孵育细胞，在37℃1小时。注：ZIF域进入细胞公关imarily通过巨吞饮，34是能量依赖性机制。因此，细胞必须在37℃下孵育高效蛋白质内化。 </li><li>从细胞用500μl钙和无镁的除去介质和洗涤三次Dulbecco氏磷酸盐缓冲盐水（DPBS），补充有肝素的0.5毫克/毫升。注意：肝素是需要除去可能复杂化下游分析表面结合的蛋白质。 </li><li> （可选）重复治疗最多三次以增强递送。 </li><li>肝素洗涤消化用0.05％胰蛋白酶-EDTA在37℃下2分钟后，立即分离处理的细胞。重悬在0.5ml DPBS中补充有1％FBS的分离的细胞。 </li><li>测量使用异硫氰酸荧光素（FITC）通道（ 图3），每个样品通过流式细胞仪35的荧光强度。调整前向散射（FSC）和侧向散射（SSC）放置的人口息上的规模，确保种群具有不同性质彼此解决。 </li><li>收集万实况事件对于每个样品，并分析利用流式细胞仪从与ZIF-EmGFP处理以那些只带有SFM处理的细胞的HeLa细胞的数据分析软件。36标准化荧光的数据。 </li></ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basics+of+flow+cytometry.">Basics of flow cytometry.</a> Methods Mol. Biol. 91, 1-24 (1998).</li><li>Mercer, A. C., Gaj, T., Sirk, S. J., Lamb, B. M., Barbas, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+endogenous+human+gene+expression+by+ligand-inducible+TALE+transcription+factors.">Regulation of endogenous human gene expression by ligand-inducible TALE transcription factors.</a> ACS Synth. Biol. 3, 723-730 (2014).</li><li>Segal, D. J., Crotty, J. W., Bhakta, M. S., Barbas, C. F., Horton, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+Aart,+a+designed+six-finger+zinc+finger+peptide,+bound+to+DNA.">Structure of Aart, a designed six-finger zinc finger peptide, bound to DNA.</a> J. Mol. Biol. 363, 405-421 (2006).</li></ol>

作者什么都没有透露。

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...

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&gt;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。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>XmaI</td>
			<td>New England Biolabs</td>
			<td>R0180L</td>
			<td></td>
		</tr>
		<tr>
			<td>SacI</td>
			<td>New England Biolabs</td>
			<td>R0156L</td>
			<td></td>
		</tr>
		<tr>
			<td>Expand High Fidelity PCR system</td>
			<td>Roche</td>
			<td>11759078001</td>
			<td></td>
		</tr>
		<tr>
			<td>dNTPs</td>
			<td>New England Biolabs</td>
			<td>N0446S</td>
			<td></td>
		</tr>
		<tr>
			<td>4-20% Tris-Glycine Mini protein gels, 1.5 mm, 10 wells</td>
			<td>Life Technologies</td>
			<td>EC6028BOX</td>
			<td></td>
		</tr>
		<tr>
			<td>2x Laemmli Sample Buffer</td>
			<td>BioRad</td>
			<td>161-0737</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 DNA Ligase</td>
			<td>Life Technologies</td>
			<td>15224-017</td>
			<td></td>
		</tr>
		<tr>
			<td>BL21 (DE3) Competent E. coli</td>
			<td>New England Biolabs</td>
			<td>C2527I</td>
			<td></td>
		</tr>
		<tr>
			<td>IPTG</td>
			<td>Thermo Scientific</td>
			<td>R0391</td>
			<td></td>
		</tr>
		<tr>
			<td>Zinc Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>208086-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Kanamycin Sulfate</td>
			<td>Fisher Scientific</td>
			<td>BP906-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G8270-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris Base</td>
			<td>Fisher Scientific</td>
			<td>BP152-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S9888-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Fisher Scientific</td>
			<td>PR-V3151&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>PMSF</td>
			<td>Thermo Scientific</td>
			<td>36978</td>
			<td></td>
		</tr>
		<tr>
			<td>Ni-NTA Agarose Resin</td>
			<td>QIAGEN</td>
			<td>30210</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G5516-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>Sigma-Aldrich</td>
			<td>I5513-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon Ultra-15 Centrifugal Filter Units</td>
			<td>EMO Millipore</td>
			<td>UFC900324</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Life Technologies</td>
			<td>11966-025</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Life Technologies</td>
			<td>10437-028</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic&#160;</td>
			<td>Life Technologies</td>
			<td>15240-062</td>
			<td></td>
		</tr>
		<tr>
			<td>24-Well Flat Bottom Plate</td>
			<td>Sigma-Aldrich</td>
			<td>CLS3527-100EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-Lysine</td>
			<td>Sigma-Aldrich</td>
			<td>P7280</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS, No Calcium, No Magnesium</td>
			<td>Life Technologies</td>
			<td>21600010</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparan Sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>H4777</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Life Technologies</td>
			<td>25300054</td>
			<td></td>
		</tr>
		<tr>
			<td>Hela cells</td>
			<td>ATCC</td>
			<td>CCL-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Nano Drop ND-1000 spectrophotometer&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAquick PCR Purification Kit</td>
			<td>QIAGEN</td>
			<td>28104</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAquick Gel Extraction Kit</td>
			<td>QIAGEN</td>
			<td>28704</td>
			<td></td>
		</tr>
	</tbody>
</table>

direct protein delivery to mammalian cells using cell-permeable cys2-his2 zinc-finger domains

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.

二指ZIF-EmGFP融合蛋白可以在大肠杆菌中表达大肠杆菌具有&gt; 95％均一性和高的产率（&gt; 25毫克/毫升）（ 图2）。在一般情况下，一个和两个手指ZIF融合蛋白可在数量几乎相同的那些野生型未修饰蛋白的制备。然而，在某些情况下，五元和六手指ZIF融合蛋白不能在产量为下游应用高至足以进行制造。 在37℃下直接应用二指ZIF-EmGFP蛋白到HeLa细胞90分钟?...

Watch this Scientific Journal Video about Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains at JoVE.com

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

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。

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&gt; 2</sub&gt; -His<sub&gt; 2</sub&gt;锌指域

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 ...

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Biology

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

9.8K 次观看.  The Scripps Research Institute. 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。 