Name: direct protein delivery to mammalian cells using cell-permeable cys2-his2 zinc-finger domains
Uploaded: 2015-03-25T15:00:00.000+00:00
Duration: 11 min 24 s
Description: Watch this Scientific Journal Video about Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains at JoVE.com

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., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+class+of+human+proteins+that+deliver+functional+proteins+into+mammalian+cells+in+vitro+and+in+vivo.">A class of human proteins that deliver functional proteins into mammalian cells in vitro and in vivo.</a> Chem. Biol. 18, 833-838 (2011).</li><li>Gaj, T., Liu, J., Anderson, K. E., Sirk, S. J., Barbas, C. F., 3rd, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+delivery+using+Cys2-His2+zinc-finger+domains.">Protein delivery using Cys2-His2 zinc-finger domains.</a> ACS Chem. Biol. 9, 1662-1667 (2014).</li><li>Radcliff, G., Jaroszeski, M. 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><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 &lt;em&gt;E. coli&lt;/em&gt;</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&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td></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 ...

direct-protein-delivery-to-mammalian-cells-using-cell-permeable-cys2

Research

JoVE Journal

Biology

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

9.9K 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。 

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

Peptide-derived Method to Transport Genes and Proteins Across Cellular and Organellar Barriers in Plants

The capacity to introduce exogenous proteins and express (or down-regulate) specific genes in plants provides a powerful tool for fundamental research as well as new applications in the field of plant biotechnology. Viable methods that currently exist for protein or gene transfer into plant cells, namely Agrobacterium and microprojectile bombardment, have disadvantages of low transformation frequency, limited host range, or a high cost of equipment and microcarriers. The following protocol outlines a simple and versatile method, which employs rationally-designed peptides as delivery agents for a variety of nucleic acid- and protein-based cargoes into plants. Peptides are selected as tools for development of the system due to their biodegradability, reduced size, diverse and tunable properties as well as the ability to gain intracellular/organellar access. The preparation, characterization and application of optimized formulations for each type of the wide range of delivered cargoes (plasmid DNA, double-stranded DNA or RNA, and protein) are described. Critical steps within the protocol, possible modifications and existing limitations of the method are also discussed.

Existing methods for the modification of plants have limited applicability. The novel peptide-derived technology proposed here promises both simplicity and efficiency in the introduction of exogenous protein or genes into the desired intracellular compartments of intact plants.

肽衍生的方法在运输跨越植物细胞的基因和蛋白质和细胞器障碍

The capacity to introduce exogenous proteins and express (or down-regulate) specific genes in plants provides a powerful tool for fundamental research as ...

科研

遗传学

Biotinylated Cell-penetrating Peptides to Study Intracellular Protein-protein Interactions

Here we present a protocol to study intracellular protein-protein interactions that is based on the widely used biotin-avidin pull-down system. The modification presented includes the combination of this technique with cell-penetrating sequences. We propose to design cell-penetrating baits that can be incubated with living cells instead of cell lysates and therefore the interactions found will reflect those that occur within the intracellular context. Connexin43 (Cx43), a protein that forms gap junction channels and hemichannels is down-regulated in high-grade gliomas. The Cx43 region comprising amino acids 266-283 is responsible for the inhibition of the oncogenic activity of c-Src in glioma cells. Here we use TAT as the cell-penetrating sequence, biotin as the pull-down tag and the region of Cx43 comprised between amino acids 266-283 as the target to find intracellular interactions in the hard-to-transfect human glioma stem cells. One of the limitations of the proposed method is that the molecule used as bait could fail to fold properly and, consequently, the interactions found could not be associated with the effect. However, this method can be especially interesting for the interactions involved in signal transduction pathways because they are usually carried out by intrinsically disordered regions and, therefore, they do not require an ordered folding. In addition, one of the advantages of the proposed method is that the relevance of each residue on the interaction can be easily studied. This is a modular system; therefore, other cell-penetrating sequences, other tags, and other intracellular targets can be employed. Finally, the scope of this protocol is far beyond protein-protein interaction because this system can be applied to other bioactive cargoes such as RNA sequences, nanoparticles, viruses or any molecule that can be transduced with cell-penetrating sequences and fused to pull-down tags to study their intracellular mechanism of action.

This is a protocol to study intracellular protein-protein interactions based on the biotin-avidin pull-down system with the novelty of combining cell-penetrating sequences. The main advantage is that the target sequence is incubated with living cells instead of cell lysates and therefore the interactions will occur within the cellular context.

化细胞穿透肽研究胞内蛋白-蛋白质相互作用

Here we present a protocol to study intracellular protein-protein interactions that is based on the widely used biotin-avidin pull-down system. The ...

生物化学

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This technology has shown potential in addressing previously challenging applications; including, delivery to primary immune cells, cell reprogramming, carbon nanotube, and quantum dot delivery. This vector-free microfluidic platform relies on mechanical disruption of the cell membrane to facilitate cytosolic delivery of the target material. Herein, we describe the detailed method of use for these microfluidic devices including, device assembly, cell preparation, and system operation. This delivery approach requires a brief optimization of device type and operating conditions for previously unreported applications. The provided instructions are generalizable to most cell types and delivery materials as this system does not require specialized buffers or chemical modification/conjugation steps. This work also provides recommendations on how to improve device performance and trouble-shoot potential issues related to clogging, low delivery efficiencies, and cell viability.

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This protocol provides detailed steps on how to use the system for a broad range of applications.

电池挤压作为一种强大的，微流控细胞内传送平台

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This ...

生物工程

Engineering Intracellular Protein Sensors in Mammalian Cells

Proteins can function as biomarkers of pathological conditions, such as neurodegenerative diseases, infections or metabolic syndromes. Engineering cells to sense and respond to these biomarkers may help the understanding of molecular mechanisms underlying pathologies, as well as to develop new cell-based therapies. While several systems that detect extracellular proteins have been developed, a modular framework that can be easily re-engineered to sense different intracellular proteins was missing.
Here, we describe a protocol to implement a modular genetic platform that senses intracellular proteins and activates a specific cellular response. The device operates on intracellular antibodies or small peptides to sense with high specificity the protein of interest, triggering the transcriptional activation of output genes, through a TEV protease (TEVp)-based actuation module. TEVp is a viral protease that selectively cleaves short cognate peptides and is widely used in biotechnology and synthetic biology for its high orthogonality to the cleavage site. Specifically, we engineered devices that recognize and respond to protein-biomarkers of viral infections and genetic diseases, including mutated huntingtin, NS3 serine-protease, Tat and Nef proteins to detect Huntington&#8217;s disease, hepatitis C virus (HCV) and human immunodeficiency virus (HIV) infections, respectively. Importantly, the system can be hand tailored for the desired input-output functional outcome, such as fluorescent readouts for biosensors, stimulation of antigen presentation for immune response, or initiation of apoptosis to eliminate unhealthy cells.

Here, we present a protocol for engineering genetically-encoded intracellular protein sensor-actuator(s). The device specifically detects target proteins through intracellular antibodies (intrabodies) and responds by switching on gene transcriptional output. A general framework is built to rapidly replace intrabodies, enabling rapid detection of any desired protein, without altering the general architecture.

Proteins can function as biomarkers of pathological conditions, such as neurodegenerative diseases, infections or metabolic syndromes. Engineering cells ...

Delivery of Proteins, Peptides or Cell-impermeable Small Molecules into Live Cells by Incubation with the Endosomolytic Reagent dfTAT

Macromolecular delivery strategies typically utilize the endocytic pathway as a route of cellular entry. However, endosomal entrapment severely limits the efficiency with which macromolecules penetrate the cytosolic space of cells. Recently, we have circumvented this problem by identifying the reagent dfTAT, a disulfide bond dimer of the peptide TAT labeled with the fluorophore tetramethylrhodamine. We have generated a fluorescently labeled dimer of the prototypical cell-penetrating peptide (CPP) TAT, dfTAT, which penetrates live cells and reaches the cytosolic space of cells with a particularly high efficiency. Cytosolic delivery of dfTAT is achieved in multiple cell lines, including primary cells. Moreover, delivery does not noticeably impact cell viability, proliferation or gene expression. dfTAT can deliver small molecules, peptides, antibodies, biologically active enzymes and a transcription factor. In this report, we describe the protocols involved in dfTAT synthesis and cellular delivery. The manuscript describes how to control the amount of protein delivered to the cytosolic space of cells by varying the amount of protein administered extracellularly. Finally, the current limitations of this new technology and steps involved in validating delivery are discussed. The described protocols should be extremely useful for cell-based assays as well as for the ex vivo manipulation and reprogramming of cells.

We describe how to deliver proteins and cell-impermeable small molecules into cultured mammalian cells by a simple co-incubation protocol with a reagent that causes endocytic organelles to become leaky.

交货蛋白质，多肽或细胞不可渗透的小分子进入活细胞被孵化与核内体溶解试剂dfTAT

Macromolecular delivery strategies typically utilize the endocytic pathway as a route of cellular entry. However, endosomal entrapment severely limits the ...

Engineering Cell-permeable Protein

The protein transduction technique enables the direct delivery of biologically active material into mammalian cells [for review see 1,2]. For this one can make use of the translocating ability of so-called cell penetrating peptides (CPPs), also designated as protein transduction domains (PTDs). The TAT-CPP derived from the human immunodeficiency virus type 1 (HIV-1) Tat (trans-activator of transcription) protein has been widely used. The positively charged TAT promotes cell permeability thereby overcoming the barriers of the cellular membrane by endocytosis or/and direct membrane penetration2. In combination with a nuclear localization signal (NLS) fusion proteins are able to enter the nucleus exhibiting functionality. Our video presentation demonstrates, as an exemplification for the engineering of cell-permeable proteins, the construction, production and application of a cell-permeable version of the DNA-modifying enzyme Cre.
Cre is a site-specific recombinase that is able to recognize and recombine 34 base pair loxP sites in mammalian cells in vitro and in vivo. Therefore the Cre/loxP system is widely used to conditionally induce mutations in the genome of living cells3,4. The delivery of active Cre recombinase to cells, however, represents a limitation.
We describe the pSESAME vector system, which allows a direct insertion of the gene-of-interest and provides a platform to rapidly clone different domains and tags used within the vector in a convenient and standardized manner. Rearranging of the different tags has been shown to modify the biochemical properties of the fusion proteins providing a possibility to achieve higher yield and better solubility. We demonstrate how to express and purify recombinant cell-permeant proteins in and from E. coli. The functionality of the recombinant Cre protein is finally validated in cell culture by assessing its intracellular recombinase activity.

Protein transduction enables the direct delivery of biologically active proteins into cells. In contrast to conventional methods such as DNA transfection or viral transduction this non-invasive paradigm allows highly efficient cellular manipulation in a titratable manner circumventing cellular toxicity and the risk of oncogenic transformation by permanent genetic modification.

工程细胞透水蛋白

The protein transduction technique enables the direct delivery of biologically active material into mammalian cells [for review see 1,2]. For this one can ...

生物学

A Convenient and General Expression Platform for the Production of Secreted Proteins from Human Cells

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. However, prokaryotic hosts are often not as appropriate for expression of human, viral or eukaryotic proteins due to toxicity of the foreign macromolecule, differences in the protein folding machinery, or due to the lack of particular co- or post-translational modifications in bacteria. Expression systems based on yeast (P. pastoris or S. cerevisiae) 1,2, baculovirus-infected insect (S. frugiperda or T. ni) cells 3, and cell-free in vitro translation systems 2,4 have been successfully used to produce mammalian proteins. Intuitively, the best match is to use a mammalian host to ensure the production of recombinant proteins that contain the proper post-translational modifications. A number of mammalian cell lines (Human Embryonic Kidney (HEK) 293, CV-1 cells in Origin carrying the SV40 larget T-antigen (COS), Chinese Hamster Ovary (CHO), and others) have been successfully utilized to overexpress milligram quantities of a number of human proteins 5-9. However, the advantages of using mammalian cells are often countered by higher costs, requirement of specialized laboratory equipment, lower protein yields, and lengthy times to develop stable expression cell lines. Increasing yield and producing proteins faster, while keeping costs low, are major factors for many academic and commercial laboratories.
Here, we describe a time- and cost-efficient, two-part procedure for the expression of secreted human proteins from adherent HEK 293T cells. This system is capable of producing microgram to milligram quantities of functional protein for structural, biophysical and biochemical studies. The first part, multiple constructs of the gene of interest are produced in parallel and transiently transfected into adherent HEK 293T cells in small scale. The detection and analysis of recombinant protein secreted into the cell culture medium is performed by western blot analysis using commercially available antibodies directed against a vector-encoded protein purification tag. Subsequently, suitable constructs for large-scale protein production are transiently transfected using polyethyleneimine (PEI) in 10-layer cell factories. Proteins secreted into litre-volumes of conditioned medium are concentrated into manageable amounts using tangential flow filtration, followed by purification by anti-HA affinity chromatography. The utility of this platform is proven by its ability to express milligram quantities of cytokines, cytokine receptors, cell surface receptors, intrinsic restriction factors, and viral glycoproteins. This method was also successfully used in the structural determination of the trimeric ebolavirus glycoprotein 5,10.
In conclusion, this platform offers ease of use, speed and scalability while maximizing protein quality and functionality. Moreover, no additional equipment, other than a standard humidified CO2 incubator, is required. This procedure may be rapidly expanded to systems of greater complexity, such as co-expression of protein complexes, antigens and antibodies, production of virus-like particles for vaccines, or production of adenoviruses or lentiviruses for transduction of difficult cell lines.

In the post-human genomics era, the availability of recombinant proteins in native conformations is crucial to structural, functional and therapeutic research and development. Here, we describe a test- and large-scale protein expression system in human embryonic kidney 293T cells that can be used to produce a variety of recombinant proteins.

一个从人体细胞分泌蛋白生产的方便，一般的表达平台

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. ...

Recombinant Protein Expression for Structural Biology in HEK 293F Suspension Cells: A Novel and Accessible Approach

The expression and purification of large amounts of recombinant protein complexes is an essential requirement for structural biology studies. For over two decades, prokaryotic expression systems such as E. coli have dominated the scientific literature over costly and less efficient eukaryotic cell lines. Despite the clear advantage in terms of yields and costs of expressing recombinant proteins in bacteria, the absence of specific co-factors, chaperones and post-translational modifications may cause loss of function, mis-folding and can disrupt protein-protein interactions of certain eukaryotic multi-subunit complexes, surface receptors and secreted proteins. The use of mammalian cell expression systems can address these drawbacks since they provide a eukaryotic expression environment. However, low protein yields and high costs of such methods have until recently limited their use for structural biology. Here we describe a simple and accessible method for expressing and purifying milligram quantities of protein by performing transient transfections of suspension grown HEK (Human Embryonic Kidney) 293F cells.

The expression of recombinant proteins by mammalian systems is becoming an attractive method for producing protein complexes for structural biology. Here we present a simple yet highly efficient expression system using suspension grown mammalian cells to purify protein complexes for structural studies.

重组蛋白表达的结构生物学在HEK 293F悬浮细胞:一种新的和可访问方法

The expression and purification of large amounts of recombinant protein complexes is an essential requirement for structural biology studies. For over two ...

A High Yield and Cost-efficient Expression System of Human Granzymes in Mammalian Cells

When cytotoxic T lymphocytes (CTL) or natural killer (NK) cells recognize tumor cells or cells infected with intracellular pathogens, they release their cytotoxic granule content to eliminate the target cells and the intracellular pathogen. Death of the host cells and intracellular pathogens is triggered by the granule serine proteases, granzymes (Gzms), delivered into the host cell cytosol by the pore forming protein perforin (PFN) and into bacterial pathogens by the prokaryotic membrane disrupting protein granulysin (GNLY). To investigate the molecular mechanisms of target cell death mediated by the Gzms in experimental in-vitro settings, protein expression and purification systems that produce high amounts of active enzymes are necessary. Mammalian secreted protein expression systems imply the potential to produce correctly folded, fully functional protein that bears posttranslational modification, such as glycosylation. Therefore, we used a cost-efficient calcium precipitation method for transient transfection of HEK293T cells with human Gzms cloned into the expression plasmid pHLsec. Gzm purification from the culture supernatant was achieved by immobilized nickel affinity chromatography using the C-terminal polyhistidine tag provided by the vector. The insertion of an enterokinase site at the N-terminus of the protein allowed the generation of active protease that was finally purified by cation exchange chromatography. The system was tested by producing high levels of cytotoxic human Gzm A, B and M and should be capable to produce virtually every enzyme in the human body in high yields.

We describe here a cost-efficient granzyme expression system using HEK293T cells that produces high yields of pure, fully glycosylated and enzymatically active protease.

人类颗粒酶在哺乳动物细胞中高产量和经济高效表达系统

When cytotoxic T lymphocytes (CTL) or natural killer (NK) cells recognize tumor cells or cells infected with intracellular pathogens, they release their ...

Efficient Mammalian Cell Expression and Single-step Purification of Extracellular Glycoproteins for Crystallization

Production of secreted mammalian proteins for structural and biophysical studies can be challenging, time intensive, and costly. Here described is a time and cost efficient protocol for secreted protein expression in mammalian cells and one step purification using nickel affinity chromatography. The system is based on large scale transient transfection of mammalian cells in suspension, which greatly decreases the time to produce protein, as it eliminates steps, such as developing expression viruses or generating stable expressing cell lines. This protocol utilizes cheap transfection agents, which can be easily made by simple chemical modification, or moderately priced transfection agents, which increase yield through increased transfection efficiency and decreased cytotoxicity. Careful monitoring and maintaining of media glucose levels increases protein yield. Controlling the maturation of native glycans at the expression step increases the final yield of properly folded and functional mammalian proteins, which are ideal properties to pursue X-ray crystallography. In some cases, single step purification produces protein of sufficient purity for crystallization, which is demonstrated here as an example case.

This is a quick, cost-efficient protocol for the production of secreted, glycosylated mammalian proteins and subsequent single-step purification with sufficient yields of homogenous protein for X-ray crystallography and other biophysical studies.

高效的哺乳动物细胞表达和细胞外糖蛋白的结晶一步纯化

Production of secreted mammalian proteins for structural and biophysical studies can be challenging, time intensive, and costly. Here described is a time ...

蛋白质直接送货到哺乳动物细胞利用细胞渗透半胱氨酸

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