Name: a convenient and general expression platform for the production of secreted proteins from human cells
Uploaded: 2012-07-31T13:00:00
Description: Watch this Scientific Journal Video about A Convenient and General Expression Platform for the Production of Secreted Proteins from Human Cells at JoVE.com

这项工作是由安大略省的艾滋病毒治疗网络研究工作津贴（的ROG-G645）和加拿大健康研究的新研究者奖（的MSH-113554）研究院JEL和多伦多大学的奖学金支持HA，FCA和纪委。笔者想感谢在斯克里普斯研究所（拉霍亚加州）马妮福斯科，Dafna先生Abelson和埃里卡Ollmann萨费尔博士提供细胞，ebolavirus; GP表达载体和一般建议。

1。准备工作 - 构造和细胞培养在开始的协议之前，应该感兴趣的基因密码子优化，在哺乳动物细胞中表达，并克隆到一个合适的表达载体，利用标准的分子生物学技术。为了确保最高的成功表达的机会，应产生多个感兴趣的基因变种。许多哺乳动物表达载体可商业化，并有各种净化标签（多聚组氨酸，血凝素，链，晕标签，谷胱甘肽S-转移，等等）。我们更倾向于使用pDISPLAY?...

<ol>
	<li>Celik, E., Calik, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+recombinant+proteins+by+yeast+cells.">Production of recombinant proteins by yeast cells.</a> Biotechnol. Adv. , (2011).</li><li>Yokoyama, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+expression+systems+for+structural+genomics+and+proteomics.">Protein expression systems for structural genomics and proteomics.</a> Curr. Opin. Chem. Biol. 7, 39-43 (2003).</li><li>Nettleship, J. E., Assenberg, R., Diprose, J. M., Rahman-Huq, N., Owens, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+the+production+of+proteins+in+insect+and+mammalian+cells+for+structural+biology.">Recent advances in the production of proteins in insect and mammalian cells for structural biology.</a> J. Struct. Biol. 172, 55-65 (2010).</li><li>Carlson, E. D., Gan, R., Hodgman, C. E., Jewett, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-free+protein+synthesis:+Applications+come+of+age.">Cell-free protein synthesis: Applications come of age.</a> Biotechnol. Adv. , (2011).</li><li>Lee, J. E. <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+the+Ebola+virus+glycoprotein+bound+to+an+antibody+from+a+human+survivor.">Structure of the Ebola virus glycoprotein bound to an antibody from a human survivor.</a> Nature. 454, 177-182 (2008).</li><li>Bowden, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+basis+of+Nipah+and+Hendra+virus+attachment+to+their+cell-surface+receptor+ephrin-B2.">Structural basis of Nipah and Hendra virus attachment to their cell-surface receptor ephrin-B2.</a> Nat. Struct. Mol. Biol. 15, 567-572 (2008).</li><li>Evans, M. J., Hartman, S. L., Wolff, D. W., Rollins, S. A., Squinto, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+expression+of+an+anti-human+C5+chimeric+Fab+utilizing+a+vector+that+replicates+in+COS+and+293+cells.">Rapid expression of an anti-human C5 chimeric Fab utilizing a vector that replicates in COS and 293 cells.</a> J. Immunol. Methods. 184, 123-138 (1995).</li><li>Ye, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-level+protein+expression+in+scalable+CHO+transient+transfection.">High-level protein expression in scalable CHO transient transfection.</a> Biotechnol. Bioeng. 103, 542-551 (2009).</li><li>Wurm, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+recombinant+protein+therapeutics+in+cultivated+mammalian+cells.">Production of recombinant protein therapeutics in cultivated mammalian cells.</a> Nat Biotechnol. 22, 1393-1398 (2004).</li><li>Lee, J. E., Fusco, M. H., Hessell, A. J., Burton, D. R., Saphire, E. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Techniques+and+tactics+used+in+determining+the+structure+of+trimeric,+prefusion+Ebola+virus+GP.">Techniques and tactics used in determining the structure of trimeric, prefusion Ebola virus GP.</a> Acta Cryst. 65, 1162-1180 (2009).</li><li>Aricescu, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eukaryotic+expression:+developments+for+structural+proteomics.">Eukaryotic expression: developments for structural proteomics.</a> Acta. Crystallogr. D Biol. Crystallogr. 62, 1114-1124 (2006).</li><li>Aricescu, A. R., Lu, W., Jones, E. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+time-+and+cost-efficient+system+for+high-level+protein+production+in+mammalian+cells.">A time- and cost-efficient system for high-level protein production in mammalian cells.</a> Acta. Crystallogr. D Biol. Crystallogr. D62, 1243-1250 (2006).</li><li>Chang, V. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycoprotein+structural+genomics:+solving+the+glycosylation+problem.">Glycoprotein structural genomics: solving the glycosylation problem.</a> Structure. 15, 267-273 (2007).</li><li>Lee, J. E., Fusco, M. H., Saphire, E. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+efficient+platform+for+screening+expression+and+crystallization+of+glycoproteins+produced+in+human+cells.">An efficient platform for screening expression and crystallization of glycoproteins produced in human cells.</a> Nature Protocols. 4, 592-604 (2009).</li><li>Nettleship, J. E., Rahman-Huq, N., Owens, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+production+of+glycoproteins+by+transient+expression+in+Mammalian+cells.">The production of glycoproteins by transient expression in Mammalian cells.</a> Methods Mol. Biol. 498, 245-263 (2009).</li><li>Neil, S. J., Zang, T., Bieniasz, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tetherin+inhibits+retrovirus+release+and+is+antagonized+by+HIV-1+Vpu.">Tetherin inhibits retrovirus release and is antagonized by HIV-1 Vpu.</a> Nature. 451, 425-430 (2008).</li><li>Nakamura, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterodimerization+of+the+IL-2+receptor+beta-+and+gamma-chain+cytoplasmic+domains+is+required+for+signalling.">Heterodimerization of the IL-2 receptor beta- and gamma-chain cytoplasmic domains is required for signalling.</a> Nature. 369, 330-333 (1994).</li><li>Wang, X., Rickert, M., Garcia, K. 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+the+quaternary+complex+of+interleukin-2+with+its+alpha,+beta,+and+gammac+receptors.">Structure of the quaternary complex of interleukin-2 with its alpha, beta, and gammac receptors.</a> Science. 310, 1159-1163 (2005).</li></ol>

10层的细胞工厂生产毫克的蛋白质数量的有效容器。使用超过其他传统的船只，如滚瓶，摇瓶或微调瓶，电池工厂的一个主要优点是，它们不需要任何额外的实验室设备的采购。一个标准的二氧化碳培养箱（6.0立方英尺）轻松容纳4个10层的细胞工厂（ 图2）。此外，这些船只需要较少的劳动比菜，瓶或滚瓶细胞和蛋白质的产生和空间;一个10层的细胞工厂，相当于使用7.5定期滚?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td>评论</td></tr><tr><td>碱性磷酸酶（BCIP / NBT）的液体底物溶液。 </td><td>西格玛</td><td> B6404 </td><td></td></tr><tr><td>抗生素/抗霉菌，100X </td><td> Invitrogen公司</td><td> 15240062 </td><td></td></tr><tr><td>抗HA的亲和力矩阵，克隆3F10 </td><td>罗氏公司</td><td> 1815016 </td><td></td></tr><tr><td>鼠单克隆抗体，抗-HA克隆16B12 </td><td> Covance公司</td><td> MMS-101P </td><td></td></tr><tr><td>细胞培养瓶的T75厘米2组织文化对待</td><td>康宁</td><td> 430641 </td><td></td></tr><tr><td>细胞培养瓶，T225厘米2组织文化对待</td><td>康宁</td><td> 431082 </td><td></td></tr><tr><td>细胞培养板,6-以及组织培养处理</td><td>康宁</td><td> 3516 </td><td></td></tr><tr><td>电池厂，10层CellSTACK的; </td><td>康宁</td><td> 3312 </td><td></td></tr><tr><td> centramate欧米茄5K盒式</td><td>鲍尔</td><td> OS005C12 </td><td></td></tr><tr><td> centramate欧米茄30K磁带</td><td>鲍尔</td><td> OS030C12 </td><td></td></tr><tr><td>玻璃层析柱，1.0×10厘米</td><td> kontes </td><td> 4204001010 </td><td></td></tr><tr><td>环丙沙星</td><td>西格玛</td><td> 17850 </td><td></td></tr><tr><td>的CO 2 </td><td></td><td></td><td></td></tr><tr><td>杜尔贝科的改良Eagle的媒体（DMEM培养基） </td><td>西格玛</td><td> D5796 </td><td></td></tr><tr><td>胎牛血清（FBS），热灭活</td><td> Invitrogen公司</td><td> 12484-028 </td><td></td></tr><tr><td> FuGENE的HD转染试剂</td><td> Promega公司</td><td> 4709691001 </td><td></td></tr><tr><td> GeneJuice转染试剂</td><td> EMD /默克</td><td> 70967-6 </td><td></td></tr><tr><td>甘氨酸</td><td>西格玛</td><td> G8898 </td><td></td></tr><tr><td>山羊抗鼠IgG F（AB&#39;）2碱性</td><td> Thermo Scientific的</td><td> 31324 </td><td></td></tr><tr><td>磷酸酶抗体</td><td></td><td></td><td></td></tr><tr><td>血凝素（HA）肽，100毫克</td><td>金斯瑞</td><td>定制合成</td><td></td></tr><tr><td> （序列YPYDVPDYA纯度为95％） </td><td></td><td></td><td></td></tr><tr><td> HEK 293T细胞</td><td> ATCC </td><td> CRL-11268 </td><td></td></tr><tr><td> householð漂白剂（4％W / V次氯酸钠） </td><td>各种品牌</td><td></td><td></td></tr><tr><td>的Immobilon - P PVDF膜</td><td> Millipore公司</td><td> IPVH07850 </td><td></td></tr><tr><td>小量质粒纯化试剂盒，快速PureLink </td><td> Invitrogen公司</td><td> K2100-11 </td><td></td></tr><tr><td> MaxiPrep质粒纯化试剂盒，PureLink HiPure </td><td> Invitrogen公司</td><td> K2100-07 </td><td></td></tr><tr><td>南三 </td><td>西格玛</td><td> S8032 </td><td></td></tr><tr><td> pDISPLAY表达载体; </td><td> Invitrogen公司</td><td> V660-20 </td><td></td></tr><tr><td>青霉素/链霉素（笔/链球菌），100X </td><td> Invitrogen公司</td><td> 15140-122 </td><td></td></tr><tr><td>磷酸盐缓冲液（PBS），无菌1X </td><td>西格玛</td><td> D8537 </td><td></td></tr><tr><td>聚乙烯亚胺（PEI），线性25 kDa的</td><td> polyscience </td><td> 23966 </td><td></td></tr><tr><td>脱脂牛奶干粉</td><td>康乃馨</td><td></td><td></td></tr><tr><td> stericup-GP PES真空过滤装置， </td><td> Millipore公司</td><td> SCGPU05RE </td><td></td></tr><tr><td> 0.22微米，500毫升容量</td><td></td><td></td><td></td></tr><tr><td>台盼蓝</td><td> Invitrogen公司</td><td> 15250061 </td><td></td></tr><tr><td>胰蛋白酶-EDTA，0.05％（W / V） </td><td> Invitrogen公司</td><td> 25300-054 </td><td></td></tr><tr><td>吐温20 </td><td>西格玛</td><td> P7949 </td><td></td></tr><tr><td>丙戊酸</td><td>西格玛</td><td> P4543 </td><td></td></tr><tr><td> Centramate切向流系统</td><td>鲍尔</td><td></td><td></td></tr><tr><td> CO 2培养箱孵化器，标准的6.0立方米。英尺。 </td><td>各种品牌</td><td></td><td></td></tr><tr><td>电泳和转印装置</td><td>各种品牌</td><td></td><td></td></tr><tr><td>孵化器，37℃ </td><td>各种品牌</td><td></td><td></td></tr></tbody></table>

a convenient and general expression platform for the production of secreted proteins from human cells

重组蛋白在细菌中表达，通常大肠杆菌大肠杆菌 ，一直毫克的蛋白质数量表达的最成功的战略。然而，原核主机通常是不为人类，病毒或真核蛋白质由于外国高分子毒性，在蛋白质折叠机制的差异，或因缺乏特别合作或翻译后的修改，在细菌的适当表达。基于酵母（ 毕赤酵母或酵母 ）1,2，杆状病毒感染的昆虫（S.秋行军虫或 T 妮 ）细胞3，和无细胞体外翻译系统的表达系统2,4已被成功地用于生产哺乳动物蛋白质。直观，最匹配的是，使用哺乳动物的主机，以确保包含正确的翻译后修饰的重组蛋白的生产。一些哺乳动物细胞株人类胚胎小子（滋肾（HEK）293，C V-1细胞在 O rigin携带的 S V40 larget T抗原（COS）中国仓鼠卵巢（CHO），和其他人）已成功地利用过度毫克数量的人体蛋白质数量5-9。然而，使用哺乳动物细胞中的优势往往成本较高，专业实验室设备的需求，降低蛋白质产量，和漫长的时间发展稳定表达细胞株的反击。提高产量和生产蛋白质的速度，同时保持成本低，是许多学术和商业实验室的主要因素。 在这里，我们描述了时间和成本效益，由两部分组成，附着HEK 293T细胞分泌的人类蛋白质表达过程。这个系统是能够生产微克到毫克数量的功能蛋白质结构，生物物理和生物化学研究。第一部分，是产生多个感兴趣的基因结构并行和瞬时转成小规模的附着的HEK 293T细胞。重组蛋白分泌到细胞培养液中的检测和分析，进行印迹分析，使用市售的抗体对矢量编码的蛋白质纯化标签指示。随后，适合大规模蛋白质生产结构瞬时转染使用聚乙烯亚胺（PEI）在10层的细胞工厂。成升体积的条件培养基中分泌蛋白集中到管理大量使用切向流过滤，净化，抗HA的亲和层析。这个平台的效用证明毫克量的细胞因子，细胞因子受体，细胞表面的受体，内在的制约因素，与病毒糖蛋白的表达能力。这种方法也被成功地用于三聚ebolavirus糖5,10结构的决心。 培养箱 ，是必需的。这个过程可能会迅速扩大到更复杂的系统，如蛋白质复合物，抗原和抗体，疫苗生产的病毒样颗粒，或生产困难的细胞株转导的腺病毒或慢病毒表达。

Watch this Scientific Journal Video about A Convenient and General Expression Platform for the Production of Secreted Proteins from Human Cells at JoVE.com

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

在人类后基因组时代，在本机构的重组蛋白的结构，功能和治疗的研究和发展是至关重要的。在这里，我们描述了测试和大规模的蛋白质在人类胚胎肾细​​胞293T，可用于生产各种重组蛋白表达系统。

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. ...

Research

JoVE Journal

Biology

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

人类胚胎干细胞（ES细胞）的繁殖

科研

生物学

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

主电穿孔的小鼠骨髓造血细胞培养制备

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

无有机溶剂和醋酸，蛋白质与考马斯亮蓝G - 250凝胶染色

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

使用基因脉冲MXcell电穿孔系统主细胞的转染效率高

The MultiBac Protein Complex Production Platform at the EMBL

在EMBL的MultiBac蛋白质复杂的生产平台

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...

Efficient Agroinfiltration of Plants for High-level Transient Expression of Recombinant Proteins

高级别瞬态表达重组蛋白的植物高效的农杆菌

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing ...

Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies

高效的生产和纯化重组小鼠Kindlin-3从昆虫细胞生物物理研究

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

膜蛋白的绿色荧光蛋白为基础的表达筛选<em&gt;大肠杆菌</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

Enrichment of Extracellular Matrix Proteins from Tissues and Digestion into Peptides for Mass Spectrometry Analysis

从富集组织及消化细胞外基质蛋白的成肽质谱分析

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of ...

High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension

重组人蛋白与HEK293悬浮细胞的瞬时转染高产表达

The art of producing recombinant proteins with complex post-translational modifications represents a major challenge for studies of structure and ...