This work was supported by grants from the Novartis Foundation for Medical-Biological Research and from the Research Pool of the University of Fribourg (to MW). We thank Li Zhao, Zhan Xu, and Solange Kharoubi Hess for technical support, as well as Radu Aricescu and Yvonne Jones (Oxford University, UK) for providing the pHLsec plasmid, and Thomas Sch&#252;rpf (Harvard Medical School) for helpful discussions.

1.生产表达质粒pHLsec-GZM的<ol><li>从人NK细胞制备总RNA用合适的RNA分离的方法和使用第一链cDNA合成试剂盒反转录（在26或NK细胞系的NK-92表达所有五个人类Gzms制备为原代细胞），继manufacturer`的建议。使用GZM基因PCR和克隆到pHLsec在23（拉杜Aricescu和伊冯·琼斯，英国牛津大学样的礼物）描述使用AGEI和KpnI位点（ 图1）放大。 注意：使用在表1所示的以下引物。 </li><li>确认无误后插入测序。展开DH5α细胞中的表达质粒和净化使用无内毒素质粒提取试剂盒，并按照制造商的说明。 </li><li>重悬在无内毒素的无菌水的纯化的质粒以2mg / ml和商店的浓度在-20℃until采用。 </li></ol> Gzms在HEK293T细胞2.表达<ol><li>试剂的制备<ol><li>制备标准培养基。以Dulbecco氏改良的Eagle培养基（DMEM）含有高葡萄糖（25毫米），Glutamax的（4毫摩尔），丙酮酸钠（1mM的）添加标准胎牛血清（FCS），青霉素（100单位/ ml），链霉素10％（ 100微克/毫升）。 </li><li>准备转染培养基。而不青霉素 - 链霉素的培养基中添加25微克/ ml的氯喹（在转染当天，从1000倍的股票在PBS，储存在-20℃下添加新鲜）。 </li><li>制备无血清培养基：向无血清培养基中的HEK293细胞中添加谷氨酰胺（4毫摩尔），青霉素（100单位/ ml），链霉素（100微克/毫升），并为2.5mg / L的两性霉素B. </li><li>准备使用前于表2和过滤器描述了一个0.45微米的过滤器的解决方案： </li></ol></li><li>扩大HEK293T细胞<ol><li>成长HEK293T细胞20毫升崇拜采用150×25mm的组织培养皿中茜。 </li><li>在80％汇合分裂细胞（分比为1：4，通常每3天）。细胞松散连接，它们可通过严格上下抽吸被机械分离的无胰蛋白酶消化。 </li><li>板的细胞转染的前一天晚上，得到60-70％汇合在转染（接种密度〜2×10 5个细胞/ ml）的一天。通常制剂规模由20至25培养皿。 </li></ol></li><li>磷酸钙转染<ol><li> 1小时转染前，改变标准培养基到20ml转染培养基。邻近的这1小时温育步骤结束时，混合400微克质粒DNA与10.95毫升双蒸2 O和在50ml试管1.55毫升2M 氯化钙 （1管/ 5菜）。 </li><li> 1至2之前转染分钟，添加12.5毫升2×HBS到12.5的DNA的钙溶液的毫升，由管倒置混合和温育30秒，在室温。 </li><li>第添加ë混合物直接加入细胞（每皿5毫升）逐滴入培养基制备2.3.2。均匀地洒在整个区域，把介质到浅橙色的颜色。 </li><li>孵育培养皿为7至11小时，在组织培养恒温箱（37℃，5％CO 2的湿润空气）。培养后，微细的析出物是可见的。移除转染介质，用预热的PBS小心冲洗并加入2.1.3制备20毫升无血清培养基中。孵育72小时在组织培养箱。 </li><li>通过运行在SDS-PAGE的细胞上清液的样品（约20微升）和用考马斯亮蓝染色来可视化颗粒酶带分析的转染和表达效率。 </li></ol></li><li>净化Gzms从培养上清镍IMAC <ol><li>培养后，弃去细胞的培养物上清液加到250ml试管并离心清晰。开展第一自旋（400 XG，10分钟，4℃C），将清除介质从分离的细胞。转移在新鲜250毫升管和自旋上清液以4000×g离心，30分钟，在4℃以除去任何残留的细胞碎片。 </li><li>加入5毫升5 M氯化钠，6.25毫升0.25M的制备NiSO 4的2M Tris碱（pH 8）中和1毫升每250毫升清除上清液。使用500ml的真空过滤器单元（0.45毫米）过滤上清。 </li><li>平衡使用合适的FPLC系统镍IMAC柱与他的结合缓冲液A（10柱体积，CV）。 </li><li>以0.5毫升/分钟，在4℃的流速施加的澄清的上清液到平衡柱。 </li><li>上清液施加之后，直到UV吸收基线达到（通常为10 CV）与他的结合缓冲液洗柱。 </li><li>洗脱Gzms具有线性20毫升梯度（0至250mM咪唑）以0.5毫升/分钟流速，同时收集2毫升的级分。分析洗脱的级分通过SDS-PAGE和考马斯染色。 </li></ol></li><li>肠激酶（EK）处理和最终的清理通过阳离子交换层析<ol><li>游泳池GZM包含在透析管≤10截留分子量组分。储存在-20℃的小样本作为预EK控制。 </li><li>在0.02单位/50毫升初始上清液添加EK直接向汇集馏分在透析管中透析O / N（至少16小时）在RT在EK-缓冲器（4L，改变缓冲一次）。 </li><li>第二天，分析EK在相比于预EK控制通过SDS-PAGE和考马斯染色处理Gzms。 </li><li>当N-末端处理完成后，改变透析缓冲至S缓冲液A（4 L）和透析另外4小时，在室温。透析过滤（0.45微米）。 </li><li>平衡一个的S-柱带有S缓冲液A与加载样品上柱以0.5ml /分钟，在4℃的流速。 </li><li>样后，直到紫外吸收基线达到（约10 CV）洗带S缓冲液A柱。洗脱Gzms用30ml的线性梯度（150〜1000毫摩尔NaCl）。 GzmAê在琵琶〜650毫米氯化钠，在GZMB〜700毫米氯化钠和GzmM在〜750毫米氯化钠。 </li><li>分析洗脱的级分通过SDS-PAGE和比色测定法（见下文）。含有Gzms和浓缩池级分（约30倍，至约100μM的浓度）的自旋过滤器（15毫升，10 MWCO）。分装浓GZM准备并储存在-80℃。 </li></ol></li></ol> 3.测试GZM活动<ol><li>试剂的制备<ol><li>制备比色测定缓冲液：50毫摩尔Tris-基地，pH为7。 <ol><li>为GzmA的测量，添加的Na-CBZ-L-赖氨酸苄硫基酯（S-BZL）（BLT）的测定缓冲液以0.2毫和5,5&#39;-二硫代双（2-硝基苯甲酸）的终浓度（DTNB），以0.22毫浓度。对于GZMB，包括0.2毫米的Boc - 丙氨酸 - 丙氨酸 - 天冬氨酸 - S-BZL（AAD）和0.22毫DTNB。对于GzmM，包括0.2毫米SUC-丙氨酸 - 丙氨酸 - 脯氨酸 - 亮氨酸对硝基苯胺（PNA）（AAPL）。合成肽是由适当的储备溶液中加入（在DMSO中，保存位置编在-20℃）到缓冲新鲜的测定之前。 </li></ol></li><li>制备裂解测定缓冲液：100mM NaCl的50毫摩尔Tris-碱，pH 7.5 </li></ol></li><li>比色法<ol><li>分发来自洗脱份小样本GZM（&lt;5μL）或个股集中在96孔平底板。包括一个阳性对照组（测试GZM制剂或粗NK细胞裂解液）和阴性对照（只缓冲）。 </li><li>加入100微升测定缓冲液到每个孔中（使用多通道移液器）。孵育10分钟，在37℃。测量的OD在酶标仪405nm处。 </li></ol></li><li>切割测定法<ol><li>为使用纯化的底物裂解测定法，共孵育的蛋白质底物（天然的或重组的，100 - 400纳米;高效GZM衬底的几个例子将在本节的末尾呈现在结果部分，并在注）与连续稀释的一个GZM在不同时间测定缓冲液（起始于400纳米）。通过SDS-PAGE和考马斯或银染色分析;或者通过免疫印迹使用特异性抗体。 </li><li>对于使用细胞裂解物裂解测定法，悬浮在1ml分析缓冲液和裂解10 6个HeLa细胞（或任何可用的肿瘤细胞系）通过在37℃下三次在乙醇/干冰浴中解冻冷冻。通过离心（在4℃下15000×g离心10分钟）除去细胞碎片。 </li><li>共孵育澄清的裂解液与Gzms各种浓度和倍以上。切割是通过使用合适的抗体进行免疫印迹检测到。 注：国际棋联基板善意的几个例子商业抗体是其中可供选择：买入（BH3相互作用域死亡激动剂）和caspase 3由GZMB 27,28切割;多个异构核核糖（hnRNPA1，A2和U）由GzmA 29切割;巨细胞病毒磷71 GzmM 30。 </li></ol></li></ol>

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P., Lieberman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alterations+in+RNA+processing+during+immune-mediated+programmed+cell+death.">Alterations in RNA processing during immune-mediated programmed cell death.</a> Proc Natl Acad Sci U S A. 109, 8688-8693 (2012).</li><li>Domselaar, R., 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=Noncytotoxic+inhibition+of+cytomegalovirus+replication+through+NK+cell+protease+granzyme+M-mediated+cleavage+of+viral+phosphoprotein+71.">Noncytotoxic inhibition of cytomegalovirus replication through NK cell protease granzyme M-mediated cleavage of viral phosphoprotein 71.</a> J Immunol. 185, 7605-7613 (2010).</li><li>Thiery, 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=Perforin+activates+clathrin-+and+dynamin-dependent+endocytosis,+which+is+required+for+plasma+membrane+repair+and+delivery+of+granzyme+B+for+granzyme-mediated+apoptosis.">Perforin activates clathrin- and dynamin-dependent endocytosis, which is required for plasma membrane repair and delivery of granzyme B for granzyme-mediated apoptosis.</a> Blood. 115, 1582-1593 (2010).</li><li>Thiery, 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=Perforin+pores+in+the+endosomal+membrane+trigger+the+release+of+endocytosed+granzyme+B+into+the+cytosol+of+target+cells.">Perforin pores in the endosomal membrane trigger the release of endocytosed granzyme B into the cytosol of target cells.</a> Nat Immunol. 12, 770-777 (2011).</li><li>Thomas, M. P., 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=Leukocyte+protease+binding+to+nucleic+acids+promotes+nuclear+localization+and+cleavage+of+nucleic+acid+binding+proteins.">Leukocyte protease binding to nucleic acids promotes nuclear localization and cleavage of nucleic acid binding proteins.</a> J Immunol. 192, 5390-5397 (2014).</li><li>Jacquemin, G., 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=Granzyme+B-induced+mitochondrial+ROS+are+required+for+apoptosis.">Granzyme B-induced mitochondrial ROS are required for apoptosis.</a> Cell death and differentiation. , (2014).</li><li>Kummer, J. A., Kamp, A. M., Citarella, F., Horrevoets, A. J., Hack, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+human+recombinant+granzyme+A+zymogen+and+its+activation+by+the+cysteine+proteinase+cathepsin+C.">Expression of human recombinant granzyme A zymogen and its activation by the cysteine proteinase cathepsin C.</a> The Journal of biological chemistry. 271, 9281-9286 (1996).</li><li>Hagn, M., Sutton, V. R., Trapani, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+colorimetric+assay+that+specifically+measures+Granzyme+B+proteolytic+activity:+hydrolysis+of+Boc-Ala-Ala-Asp-S-Bzl.">A colorimetric assay that specifically measures Granzyme B proteolytic activity: hydrolysis of Boc-Ala-Ala-Asp-S-Bzl.</a> Journal of visualized experiments : JoVE. , e52419 (2014).</li><li>Huang, M. T., Gorman, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intervening+sequences+increase+efficiency+of+RNA+3'+processing+and+accumulation+of+cytoplasmic+RNA.">Intervening sequences increase efficiency of RNA 3' processing and accumulation of cytoplasmic RNA.</a> Nucleic Acids Res. 18, 937-947 (1990).</li><li>Luthman, H., Magnusson, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+efficiency+polyoma+DNA+transfection+of+chloroquine+treated+cells.">High efficiency polyoma DNA transfection of chloroquine treated cells.</a> Nucleic Acids Res. 11, 1295-1308 (1983).</li><li>Magee, A. I., Grant, D. A., Hermon-Taylor, J., Offord, R. 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The authors have nothing to disclose.

GzmA和GZMB的经典和广泛研究的作用是诱导凋亡在哺乳动物细胞中的其胞质递送由所述孔形成蛋白PFN 1之后。最近，Gzms的细胞毒性谱显著从哺乳动物细胞扩大到细菌9,10，以及某些寄生虫11。此外，GzmA和GZMB的非经典，胞外功能以及各种孤儿Gzms的生物学意义仍不清楚。因此，Gzms的健壮时间和成本的高效表达和纯化系统中，通过该协议规定，将是对这些未来的研究有很大的帮助...

所述Gzms是一族局部存在的CTL专门溶酶体和NK细胞1高度同源的丝氨酸蛋白酶。这些杀伤细胞的细胞毒性颗粒还包含与膜破坏蛋白质PFN和GNLY被在识别去往消除2,3靶细胞的同时释放与Gzms。有五个Gzms在人类（GzmA，B，H，K和M），和10 Gzms小鼠（GzmA - G，K，M和N）。 GzmA和GZMB是最丰富的和广泛的研究在人类和小鼠1。然而，最近的研究已经开始研究细胞死亡途径以及由在健康和疾病4，另外，所谓孤儿Gzms介导的额外的生物效应。 该Gzms最著名的功能，特别是GzmA和GZMB的，是程序性细胞死亡在哺乳动物细胞中的诱导时由PFN 5,6-递送到靶细胞。然而，最近的研究还PFN 7,8展示了Gzms细胞外效应与免疫调节和炎症产生深远的影响，独立胞浆交付。这是在Gzms胞浆入境后有效地杀伤细胞的光谱也于近日从哺乳动物细胞扩大至9,10细菌，甚至某些寄生虫11。最近的这些发现开辟了一个全新的领域进行研究GZM。因此，经济高效，高产哺乳动物表达系统将显著缓解的方式为那些未来的研究。 天然人，小鼠和大鼠Gzms已成功地从CTL和NK细胞线12-14的颗粒级分纯化。然而，在我们手中的这样的纯化技术的产率小于0.1毫克/升细胞培养物（未发表的观测和12）的范围内。此外，单个GZM由诺特的色谱分离而不污染řGzms和/或蛋白质，也存在于该颗粒是具有挑战性（未公布的数据和12,14）。重组Gzms产生于细菌15，酵母16，昆虫细胞17和甚至在哺乳动物细胞如HEK 293 18,19。仅在哺乳动物表达系统承担产生重组酶具有相同于天然细胞毒性蛋白质的翻译后修饰的潜力。翻译后修饰已牵涉与通过内吞作用的特异性摄取和蛋白酶的靶细胞20-22内的细胞内定位。因此，通过使用pHLsec 23质粒骨干GZM表达（拉杜Aricescu和伊冯·琼斯，英国牛津大学一个样的礼物），我们建立了一个简单，时间和成本效益的系统，高产蛋白生产HEK293T细胞。 pHLsec结合CMV增强用鸡Β肌动蛋白启动子;在一起，这些元件demonstra泰德在各种细胞系24中的最强的启动子活性。此外，该质粒含有兔Β珠蛋白内含子，优化的Kozak和分泌信号，一个赖氨酸的6xHis标签和一个多聚A信号。插入可方便地分泌信号，并确保对缺乏适当的N-末端结构域的蛋白的最佳表达和分泌效率的赖氨酸的6xHis标签（ 图1）之间进行克隆。为Gzms的表达，我们换成由载体随后肠激酶（EK）的网站（DDDDK）提供的分泌信号内源分泌信号序列，使得EK治疗活化分泌Gzms（活性Gzms开始与N-末端氨基酸序列IIGG 25）。另外，在有利于该方法，HEK293T细胞在低价介质快速增长，如Dulbecco改进的Eagle培养基（DMEM），并且非常适合于成本效益的磷酸钙转染法。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>TRIzol Reagent</td>
			<td>Invitrogen</td>
			<td>15596-026</td>
			<td>Total RNA isolation kit</td>
		</tr>
		<tr>
			<td>SuperScript II Reverse Transcriptase</td>
			<td>Invitrogen</td>
			<td>18064-014</td>
			<td></td>
		</tr>
		<tr>
			<td>Phusion High-Fidelity DNA Polymerase</td>
			<td>NEB</td>
			<td>M0530L</td>
			<td></td>
		</tr>
		<tr>
			<td>EndoFree Plasmid Maxi Kit</td>
			<td>QIAGEN</td>
			<td>12362</td>
			<td></td>
		</tr>
		<tr>
			<td>EX-CELL 293 Serum-Free Medium for HEK 293 Cells</td>
			<td>Sigma</td>
			<td>14571C</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, high glucose, GlutaMAX Supplement, pyruvate</td>
			<td>Gibco</td>
			<td>31966-021</td>
			<td></td>
		</tr>
		<tr>
			<td>SnakeSkin Dialysis Tubing, 10K MWCO</td>
			<td>Thermo Scientific</td>
			<td>68100</td>
			<td></td>
		</tr>
		<tr>
			<td>Enterokinase from bovine intestine</td>
			<td>Sigma</td>
			<td>E4906</td>
			<td>recombinant, &#8805;20&#160;units/mg protein</td>
		</tr>
		<tr>
			<td>HisTrap Excel 5ml-column</td>
			<td>GE Healthcare</td>
			<td>17-3712-06&#160;</td>
			<td>Nickel IMAC</td>
		</tr>
		<tr>
			<td>HiTrap SP HP 5 ml-column</td>
			<td>GE Healthcare</td>
			<td>17-1152-01&#160;</td>
			<td>S column</td>
		</tr>
		<tr>
			<td>N-&#945;-Cbz-L-lysine thiobenzylester (BLT)</td>
			<td>Sigma</td>
			<td>C3647</td>
			<td>GzmA substrate</td>
		</tr>
		<tr>
			<td>Boc-Ala-Ala-Asp-S-Bzl (AAD)</td>
			<td>MP Biomedicals</td>
			<td>2193608 _10mg</td>
			<td>GzmB substrate</td>
		</tr>
		<tr>
			<td>Suc-Ala-Ala-Pro-Leu-p-nitroanilide (AAPL)</td>
			<td>Bachem</td>
			<td></td>
			<td>GzmM substrate</td>
		</tr>
		<tr>
			<td>5,5&#8242;-dithio-bis(2-nitrobenzoic acid) (DTNB)</td>
			<td>Sigma</td>
			<td>D8130</td>
			<td>Ellman`s reagent</td>
		</tr>
	</tbody>
</table>

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.

在下面的部分中，我们将提出一个GzmA制剂的一个完整的文件照亮方法。我们还成功纯化GZMB和GzmM，在生产方面的净化效率和活动类似的结果。然而，从这些准备工作后，我们将只显示几个选定的数据块。从293T细胞按照目前的协议GZMB准备在几个已发表 ​​的研究中使用，突出了他们活动的各种生物检测系统9,29,31-34。 一个典型的GzmA纯化示于图2A中 。有效?...

Watch this Scientific Journal Video about A High Yield and Cost-efficient Expression System of Human Granzymes in Mammalian Cells at JoVE.com

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

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 ...

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JoVE Journal

Biology

9.6K 次观看.  Boston Children’s Hospital and Harvard Medical School. We describe here a cost-efficient granzyme expression system using HEK293T cells that produces high yields of pure, fully glycosylated and enzymatically active protease. 