This work was supported by NIH R01-HL119813 (to T.J.B.), American Lung Association RG-196051 (to T.J.B.), a CIMED Pilot and Feasibility grant (to T.J.B.), American Heart Association Predoctoral Fellowships 14PRE19970008 (to Z.Y.) and 15PRE22110004 (to D.L.K.).

1.生产质粒DNA的毫克量为大规模瞬时转染的<ol><li>克隆感兴趣的蛋白质成使用限制性位点克隆，或其它适当的技术高的拷贝数的哺乳动物表达载体中。 <ol><li>为获得最佳的结果，使用pHLsec 7矢量，其中有一个内置的C末端的6His标签，强启动子Kozak序列和优化的分泌信号。 </li></ol></li><li>变换质粒到感受态细胞。 <ol><li>添加20微升感受态大肠杆菌细胞的到1微克质粒DNA和在冰上孵育30分钟。 </li><li>热休克的细胞在42℃下35秒，然后在冰上孵育2分钟。 </li><li>添加300μl的微生物生长培养基（SOC），并在37℃下进行45分钟，摇动以220rpm。 </li><li>板细胞在琼脂平板上适当的抗生素选择。 <ol><li>使用100微克/毫升羧苄青霉素，如果所述质粒是在pHLsec矢量。 </li></ol></li></ol></li><li>在250ml的Luria肉汤（LB）媒体的培养菌落补充有100微克/毫升抗生素（羧苄青霉素）O / N在37℃下，振荡以220rpm。 </li><li>净化从根据制造商的方案使用高速质粒Maxi试剂盒培养物DNA。 <ol><li>洗脱，代替缓冲器的TE缓冲液中的EB（10mM的三Cl，pH值8.5）的DNA。 </li><li>分装纯化的质粒在在-20℃所需的转染和存储量。 </li></ol></li></ol>的293F细胞2.大型文化和瞬时转染<ol><li>补充的1L 293F媒体用10ml谷氨酰胺和5ml青霉素/链霉素（均100倍）。保存在4℃。 5 ml的青霉素/链霉素是一个足够的强度在无血清条件下转染和在减压抗生素浓度提高细胞生存力，从而提高蛋白质的产量。 </li><li>文化293F细胞在300毫升的媒体在1升的聚碳酸酯挡板锥形瓶中通气帽s的37℃，8％CO 2中，一边摇动在标准组织培养箱中。 </li><li>稀释细胞之一转染前一天5×10 5 / ml的密度。 </li><li>转染，补充培养基中加入的2％10％体积w / v的在293F媒体电池升压的一天。 <ol><li>使用根据制造商的说明和使用补充剂根据需要以达到500毫克/分升的葡萄糖浓度的血糖监测仪测量葡萄糖浓度。 </li></ol></li><li>加kifunensine（1微克/毫升的最终浓度）在此步骤中，以控制蛋白质的糖基化。 </li><li>的DNA的计算量所需的每1×10 6个细胞1微克质粒。在无菌条件下，稀释在5ml无血清培养基中的DNA。 </li><li>计算所需的每2 1微克质粒微升转染试剂转染试剂的体积。在无菌条件下，稀转染试剂（PEI-TMC-25）在5ml无血清培养基。 </li><li>加转染试剂成在1ml的增量DNA溶液，轻轻混合。温育30分钟，在RT下试剂-DNA复合物的形成。然后将溶液添加到细胞中逐滴的方式。 </li><li>允许转染的细胞表达蛋白72-96小时。补充用〜10％的体积电池升压日常必要的媒体，或保持血糖读数400〜600毫克/分升。 </li></ol> 3.净化<ol><li>滗培养到离心瓶中，离心20分钟，1300×g离心以沉淀细胞，然后回收上清。如果需要的话，旋转的第二时间和/或使用0.22μm的过滤器，以澄清上清液。 </li><li>加10％的体积的10倍的Ni-NTA结合缓冲液（1.5M氯化钠，0.5 MK 2 HPO 4，0.1M的Tris pH值8.5，50mM咪唑）。 </li><li>通过加入2ml的Ni-NTA浆在一个柱并用1X结合缓冲液10倍柱体积（CV）的平衡准备一个重力柱。如果可能的话，请在4℃的房间内的所有列的步骤。可选择性伊利，寒意蛋白和列步骤之前，冰所有的缓冲区，并保持蛋白质和收集流过冰。 注意:的Ni-NTA浆料是50％的树脂体积和制造商的说明结合容量为50毫克/毫升。的Ni-NTA珠可以被重新充电用于多种用途的</li><li>流上清液过树脂，并收集流过。重复此步骤。 </li><li>用10CV的洗涤缓冲液洗涤（300毫摩尔NaCl，50 mM的K 2 HPO 4，20mM咪唑的pH 8）中。 </li><li>洗脱蛋白在5 CV的洗脱缓冲液（300mM NaCl的50 mM的K 2 HPO 4，250mM咪唑pH 8）中。 </li><li>如果去糖基化是必需的: <ol><li>为0.5 ml的终体积，洗脱液浓缩，用离心浓缩器0.43毫升如果沉淀的形式，通过离心沉淀的任何碎屑在16000×g离心和4℃。 </li><li>加入50微升500毫米柠檬酸钠pH值5.5。 </li><li>加入20微升EndoHf（1×10 6单位/毫升）中。在室温下孵育2小时。 不E:酶工作最佳，在37℃，这可能会导致浓缩的蛋白质聚集，延长对RT温育后，如果去糖基化，通过SDS-PAGE或免疫印迹评估，是不完整的。酶不具有在4℃下的活性。 </li><li>以除去EndoHf:洗净直链淀粉树脂3倍在磷酸盐缓冲盐水（PBS）或最终贮存缓冲液。孵育蛋白质与树脂1小时，在4℃。自旋5分钟，在1000×g离心沉淀珠粒并回收上清。 </li><li>使用适当的分子量截留离心过滤器浓缩蛋白质和缓冲液交换成存储缓冲液（150毫摩尔NaCl和20mM HEPES pH7.5）中。 </li></ol></li></ol>

<ol>
	<li>Meyer, S., 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=Multi-host+expression+system+for+recombinant+production+of+challenging+proteins.">Multi-host expression system for recombinant production of challenging proteins.</a> PLoS One. 8, e68674 (2013).</li><li>Chang, V. T., 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=Glycoprotein+structural+genomics:+solving+the+glycosylation+problem.">Glycoprotein structural genomics: solving the glycosylation problem.</a> Structure. 15, 267-273 (2007).</li><li>Rich, J. R., Withers, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+methods+for+the+production+of+homogeneous+human+glycoproteins.">Emerging methods for the production of homogeneous human glycoproteins.</a> Nat Chem Biol. 5, 206-215 (2009).</li><li>Wu, K., 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=TREM-2+promotes+macrophage+survival+and+lung+disease+after+respiratory+viral+infection.">TREM-2 promotes macrophage survival and lung disease after respiratory viral infection.</a> J Exp Med. 212, 681-697 (2015).</li><li>Yang, C., 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=Mitigated+cytotoxicity+and+tremendously+enhanced+gene+transfection+efficiency+of+PEI+through+facile+one-step+carbamate+modification.">Mitigated cytotoxicity and tremendously enhanced gene transfection efficiency of PEI through facile one-step carbamate modification.</a> Adv Healthc Mater. 2, 1304-1308 (2013).</li><li>Elbein, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycosidase+inhibitors:+inhibitors+of+N-linked+oligosaccharide+processing.">Glycosidase inhibitors: inhibitors of N-linked oligosaccharide processing.</a> FASEB J. 5, 3055-3063 (1991).</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. 62, 1243-1250 (2006).</li><li>Kelker, M. S., Debler, E. W., Wilson, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+mouse+triggering+receptor+expressed+on+myeloid+cells+1+(TREM-1)+at+1.76+A.">Crystal structure of mouse triggering receptor expressed on myeloid cells 1 (TREM-1) at 1.76 A.</a> J Mol Biol. 344, 1175-1181 (2004).</li><li>Kober, D. L., 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=Preparation,+crystallization,+and+preliminary+crystallographic+analysis+of+wild-type+and+mutant+human+TREM-2+ectodomains+linked+to+neurodegenerative+and+inflammatory+diseases.">Preparation, crystallization, and preliminary crystallographic analysis of wild-type and mutant human TREM-2 ectodomains linked to neurodegenerative and inflammatory diseases.</a> Protein Expr Purif. 96, 32-38 (2014).</li><li>Yurtsever, Z., 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=Self-cleavage+of+human+CLCA1+protein+by+a+novel+internal+metalloprotease+domain+controls+calcium-activated+chloride+channel+activation.">Self-cleavage of human CLCA1 protein by a novel internal metalloprotease domain controls calcium-activated chloride channel activation.</a> J Biol Chem. 287, 42138-42149 (2012).</li><li>Sala-Rabanal, M., Yurtsever, Z., Nichols, C. G., Brett, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secreted+CLCA1+modulates+TMEM16A+to+activate+Ca-dependent+chloride+currents+in+human+cells.">Secreted CLCA1 modulates TMEM16A to activate Ca-dependent chloride currents in human cells.</a> Elife. 4, (2015).</li><li>Niesen, F. H., Berglund, H., Vedadi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+differential+scanning+fluorimetry+to+detect+ligand+interactions+that+promote+protein+stability.">The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability.</a> Nat Protoc. 2, 2212-2221 (2007).</li></ol>

The authors declare that they have no competing financial interests.

HEK 293F细胞提供强大的生产需要翻译后修饰的蛋白质。该系统允许含有二硫化物，糖基化，和磷酸化，否则将使用更常规的表达的工具不存在本机折叠的蛋白质的快速且可伸缩的表达。此外，该系统可用于表达和多蛋白复合物的纯化只需通过共转染多种质粒的构建。此外TREM2，该系统已经被广泛地用于与感兴趣在实验室-10,11-其它蛋白质功能研究。哺乳动物细胞还提供用于体内实验?...

了解蛋白质结构在原子级别是关键揭露生物学途径和疾病的分子基础。 X射线蛋白质晶体是用于确定大分子结构最广泛使用的/适用的方法。该方法的主要挑战是获得足够数量的正确折叠的，纯的蛋白质。这变得尤其分泌哺乳动物蛋白质，其经历特定的翻译后修饰工作时的一个问题。 细菌表达的蛋白质是沉积在蛋白质数据银行1中结晶蛋白质的主要来源。细菌表达系统在很大程度上是优选的，因为它们是快速，廉价和通常产生蛋白质的产量高。然而，哺乳动物蛋白的胞外域在细菌中表达通常不正确折叠的，在所要求的情况下的重折叠和大量的纯化步骤以获得均匀FOLDED蛋白质。此外，许多哺乳动物蛋白质需要的翻译后糖基化，以达到适当的折叠2。虽然在酵母或昆虫细胞中表达和糖基化可以克服的折叠问题，翻译后修饰，包括糖基化，显著从那些哺乳动物细胞3的不同，产生具有不正确的或不均质的修饰蛋白质。 哺乳动物细胞表达的所有必需的分子机制，以确保适当的翻译后修饰和折叠;然而，这些表达系统通常不优选由大多数实验室，由于有限的产率和试剂和消耗品成本高。聚乙烯亚胺（PEI），一个标准的转染试剂是相对便宜的，但施加相当的细胞毒性和低转染效率，从而导致在细胞培养基，脱氧核糖核酸成本增加，并培养设备。许多替代PEI是贵得离谱。我们解决这些问题通过描述和化学修饰的PEI的快速和相对便宜的方法为分泌的哺乳动物蛋白质的表达，随后单步纯化的改进的细胞培养工具的组合。这种强大的方法给出了足够的产率的功能和生化研究4中，在某些情况下，导致蛋白质适合于结晶，无需进一步纯化。 本协议描述的几种技术，以最大限度地表达和产生于人类胚胎肾（HEK）分泌的哺乳动物蛋白悬浮培养细胞293F。转染效率（成本），蛋白生产和纯化都极大地按照该协议增强。 PEI通过单步开环反应中加入氨基甲酸酯改性（PEI-TMC-25，合成和性质的详细在文献5中所述 ）大大提高转染效率，从阳离子减少的细胞毒性膜破坏，并相应地降低实验成本。此外，细胞存活率和蛋白质表达大大用加入了培养补充供应葡萄糖和维生素改善。重要的是，用于生产糖基化蛋白，治疗kifunensine的，甘露糖苷酶I的无毒的化学抑制剂，产生的蛋白质与定义，未成熟的聚糖，其可以通过该糖苷内切酶EndoHf被除去，得到的蛋白质与单个N-乙酰葡糖胺代替全长N-连接多糖6。最后，蛋白质分泌到无血清，化学定义的培养基允许快速和简便的纯化用于结构和生化研究。单步镍 - 次氮基三乙酸（的Ni-NTA）树脂纯化去除大多数在上清液污染物种类和，在某些情况下，可以产生足够的纯度为蛋白质结晶。

<table>
	<tbody>
		<tr>
			<td>Culture Flasks</td>
			<td>GeneMate</td>
			<td>F-5909B</td>
			<td></td>
		</tr>
		<tr>
			<td>293 Freestyle Media</td>
			<td>Gibco/Life Technologies</td>
			<td>12338-018</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>Gibco/Life Technologies</td>
			<td>35050-061</td>
			<td>Use in place of Glutamine</td>
		</tr>
		<tr>
			<td>Hype 5 transfection reagent</td>
			<td>Oz Biosciences</td>
			<td>HY01500</td>
			<td></td>
		</tr>
		<tr>
			<td>293fectin transfection reagent</td>
			<td>Life Technologies</td>
			<td>12347019</td>
			<td></td>
		</tr>
		<tr>
			<td>PEI transfection reagent</td>
			<td>Sigma-Aldrich</td>
			<td>408727</td>
			<td></td>
		</tr>
		<tr>
			<td>Maxiprep Kit</td>
			<td>Qiagen</td>
			<td>12162</td>
			<td></td>
		</tr>
		<tr>
			<td>Ni-NTA Superflow&#160;</td>
			<td>Qiagen</td>
			<td>30430</td>
			<td></td>
		</tr>
		<tr>
			<td>Endo Hf</td>
			<td>NEB</td>
			<td>P0703L</td>
			<td></td>
		</tr>
		<tr>
			<td>Amylose Resin</td>
			<td>NEB</td>
			<td>E8021S</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Boost R05.2</td>
			<td>HyClone</td>
			<td>SH30584.02</td>
			<td>Cell Culture Supplement</td>
		</tr>
		<tr>
			<td>GlucCell</td>
			<td>CESCO Bioengineering</td>
			<td>DG2032</td>
			<td>Glucose Monitoring System</td>
		</tr>
		<tr>
			<td>Opti-MEM</td>
			<td>Life Technologies</td>
			<td>519850.91</td>
			<td>Serum Free Medium for DNA transfection</td>
		</tr>
		<tr>
			<td>Luria Broth (LB Media)</td>
			<td>Life Technologies</td>
			<td>10855-001</td>
			<td></td>
		</tr>
		<tr>
			<td>GC10 Competent Cells</td>
			<td>Sigma-Aldrich</td>
			<td>G2919</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

本文如下施加到分泌13 kDa的免疫球蛋白（Ig）的结构域，从表达的髓样细胞上2（hTREM2，残基19-132）的人蛋白质触发受体该表达系统的结果。 TREM2是包含具有两个二硫键和两个N-连接糖基化位点的胞外单个Ig结构域I型跨膜蛋白。不像许多其他的Ig结构域蛋白8，TREM2是不适合细菌包涵体9复性。随后的诱变证实N联聚糖都需要适当的表达和折叠。以促进结构和功能研究，TREM2引入pHLsec矢量?...

Watch this Scientific Journal Video about Efficient Mammalian Cell Expression and Single-step Purification of Extracellular Glycoproteins for Crystallization at JoVE.com

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

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 ...

efficient-mammalian-cell-expression-single-step-purification

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

13.7K 次观看.  Washington University School of Medicine. 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.

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

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

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

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 ...

科研

生物学

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

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

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 ...

Pulse-chase Analysis of N-linked Sugar Chains from Glycoproteins in Mammalian Cells

Attachment of the Glc3Man9GlcNAc2 precursor oligosaccharide to nascent polypeptides in the ER is a common modification for secretory proteins. Although this modification was implicated in several biological processes, additional aspects of its function are emerging, with recent evidence of its role in the production of signals for glycoprotein quality control and trafficking. Thus, phenomena related to N-linked glycans and their processing are being intensively investigated. Methods that have been recently developed for proteomic analysis have greatly improved the characterization of glycoprotein N-linked glycans. Nevertheless, they do not provide insight into the dynamics of the sugar chain processing involved. For this, labeling and pulse-chase analysis protocols are used that are usually complex and give very low yields. We describe here a simple method for the isolation and analysis of metabolically labeled N-linked oligosaccharides. The protocol is based on labeling of cells with [2-3H] mannose, denaturing lysis and enzymatic release of the oligosaccharides from either a specifically immunoprecipitated protein of interest or from the general glycoprotein pool by sequential treatments with endo H and N-glycosidase F, followed by molecular filtration (Amicon). In this method the isolated oligosaccharides serve as an input for HPLC analysis, which allows discrimination between various glycan structures according to the number of monosaccharide units comprising them, with a resolution of a single monosaccharide. Using this method we were able to study high mannose N-linked oligosaccharide profiles of total cell glycoproteins after pulse-chase in normal conditions and under proteasome inhibition. These profiles were compared to those obtained from an immunoprecipitated ER-associated degradation (ERAD) substrate. Our results suggest that most NIH 3T3 cellular glycoproteins are relatively stable and that most of their oligosaccharides are trimmed to Man9-8GlcNAc2. In contrast, unstable ERAD substrates are trimmed to Man6-5GlcNAc2 and glycoproteins bearing these species accumulate upon inhibition of proteasomal degradation.

We describe a method for analysis of the alteration of N-linked glycans through the early life of glycoproteins after their biosynthesis in mammalian cells. This is achieved by pulse-chase analysis of metabolically labeled glycans, enzymatic release from glycoproteins and examination by HPLC.

N -连接糖链的糖蛋白在哺乳动物细胞中的脉冲大通分析

Attachment of the Glc3Man9GlcNAc2 precursor oligosaccharide to nascent polypeptides in the ER is a common modification for secretory proteins. Although ...

MISSION esiRNA for RNAi Screening in Mammalian Cells

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as small interfering RNAs (siRNAs). The short double-stranded RNAs originate from longer double stranded precursors by the activity of Dicer, a protein of the RNase III family of endonucleases. The resulting fragments are components of the RNA-induced silencing complex (RISC), directing it to the cognate target mRNA. RISC cleaves the target mRNA thereby reducing the expression of the encoded protein1,2,3. RNAi has become a powerful and widely used experimental method for loss of gene function studies in mammalian cells utilizing small interfering RNAs.
Currently two main methods are available for the production of small interfering RNAs. One method involves chemical synthesis, whereas an alternative method employs endonucleolytic cleavage of target specific long double-stranded RNAs by RNase III in vitro. Thereby, a diverse pool of siRNA-like oligonucleotides is produced which is also known as endoribonuclease-prepared siRNA or esiRNA. A comparison of efficacy of chemically derived siRNAs and esiRNAs shows that both triggers are potent in target-gene silencing. Differences can, however, be seen when comparing specificity. Many single chemically synthesized siRNAs produce prominent off-target effects, whereas the complex mixture inherent in esiRNAs leads to a more specific knockdown10.
In this study, we present the design of genome-scale MISSION esiRNA libraries and its utilization for RNAi screening exemplified by a DNA-content screen for the identification of genes involved in cell cycle progression. We show how to optimize the transfection protocol and the assay for screening in high throughput. We also demonstrate how large data-sets can be evaluated statistically and present methods to validate primary hits. Finally, we give potential starting points for further functional characterizations of validated hits.

Here we use a human esiRNA library in a high-throughput screen for genes involved in cell division. We demonstrate how to set up and conduct an esiRNA screens, as well as how to analyze and validate the results.

在哺乳动物细胞的RNAi筛选使命esiRNA

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as ...

Expression Analysis of Mammalian Linker-histone Subtypes

Linker histone H1 binds to the nucleosome core particle and linker DNA, facilitating folding of chromatin into higher order structure. H1 is essential for mammalian development1 and regulates specific gene expression in vivo2-4. Among the highly conserved histone proteins, the family of H1 linker histones is the most heterogeneous group. There are 11 H1 subtypes in mammals that are differentially regulated during development and in different cell types. These H1 subtypes include 5 somatic H1s (H1a-e), the replacement H10, 4 germ cell specific H1 subtypes, and H1x5. The presence of multiple H1 subtypes that differ in DNA binding affinity and chromatin compaction ability6-9 provides an additional level of modulation of chromatin function. Thus, quantitative expression analysis of individual H1 subtypes, both of mRNA and proteins, is necessary for better understanding of the regulation of higher order chromatin structure and function.
Here we describe a set of assays designed for analyzing the expression levels of individual H1 subtypes (Figure 1). mRNA expression of various H1 variant genes is measured by a set of highly sensitive and quantitative reverse transcription-PCR (qRT-PCR) assays, which are faster, more accurate and require much less samples compared with the alternative approach of Northern blot analysis. Unlike most other cellular mRNA messages, mRNAs for most histone genes, including the majority of H1 genes, lack a long polyA tail, but contain a stem-loop structure at the 3' untranslated region (UTR)10. Therefore, cDNAs are prepared from total RNA by reverse transcription using random primers instead of oligo-dT primers. Realtime PCR assays with primers specific to each H1 subtypes (Table 1) are performed to obtain highly quantitative measurement of mRNA levels of individual H1 subtypes. Expression of housekeeping genes are analyzed as controls for normalization. 
The relative abundance of proteins of each H1 subtype and core histones is obtained through reverse phase high-performance liquid chromatography (RP-HPLC) analysis of total histones extracted from mammalian cells11-13. The HPLC method and elution conditions described here give optimum separations of mouse H1 subtypes. By quantifying the HPLC profile, we calculate the relative proportion of individual H1 subtypes within H1 family, as well as determine the H1 to nucleosome ratio in the cells.

We describe a set of assays to analyze expression levels of H1 linker histones. mRNA of individual H1 genes are quantitatively measured by random primer based reverse transcription followed by real-time PCR, whereas protein quantification of H1 histones is achieved by HPLC analysis.

哺乳动物链接组蛋白亚型表达分析

Linker histone H1 binds to the nucleosome core particle and linker DNA, facilitating folding of chromatin into higher order structure.  H1 is essential ...

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

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing worldwide demand for pharmaceuticals due to its limited scalability and high cost. Plants have shown to be one of the most promising alternative pharmaceutical production platforms that are robust, scalable, low-cost and safe. The recent development of virus-based vectors has allowed rapid and high-level transient expression of recombinant proteins in plants. To further optimize the utility of the transient expression system, we demonstrate a simple, efficient and scalable methodology to introduce target-gene containing Agrobacterium into plant tissue in this study. Our results indicate that agroinfiltration with both syringe and vacuum methods have resulted in the efficient introduction of Agrobacterium into leaves and robust production of two fluorescent proteins; GFP and DsRed. Furthermore, we demonstrate the unique advantages offered by both methods. Syringe infiltration is simple and does not need expensive equipment. It also allows the flexibility to either infiltrate the entire leave with one target gene, or to introduce genes of multiple targets on one leaf. Thus, it can be used for laboratory scale expression of recombinant proteins as well as for comparing different proteins or vectors for yield or expression kinetics. The simplicity of syringe infiltration also suggests its utility in high school and college education for the subject of biotechnology. In contrast, vacuum infiltration is more robust and can be scaled-up for commercial manufacture of pharmaceutical proteins. It also offers the advantage of being able to agroinfiltrate plant species that are not amenable for syringe infiltration such as lettuce and Arabidopsis. Overall, the combination of syringe and vacuum agroinfiltration provides researchers and educators a simple, efficient, and robust methodology for transient protein expression. It will greatly facilitate the development of pharmaceutical proteins and promote science education.

Plants offer a novel system for the production of pharmaceutical proteins on a commercial scale that is more scalable, cost-efficient and safe than current expression paradigms. In this study, we report a simple and convenient, yet scalable approach to introduce target-gene containing Agrobacterium tumefaciens into plants for protein transient expression.

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

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and the control of gene transcription in the cell nucleus. The kindlins are ~75 kDa multidomain proteins and bind to an NPxY motif and upstream T/S cluster of the integrin &#946;-subunit cytoplasmic tail. The hematopoietically-important kindlin isoform, kindlin-3, is critical for platelet aggregation during thrombus formation, leukocyte rolling in response to infection and inflammation and osteoclast podocyte formation in bone resorption. Kindlin-3&#39;s role in these processes has resulted in extensive cellular and physiological studies. However, there is a need for an efficient method of acquiring high quality milligram quantities of the protein for further studies. We have developed a protocol, here described, for the efficient expression and purification of recombinant murine kindlin-3 by use of a baculovirus-driven expression system in Sf9 cells yielding sufficient amounts of high purity full-length protein to allow its biophysical characterization. The same approach could be taken in the study of the other mammalian kindlin isoforms.

Kindlins are fundamental to cell adhesion through integrins but studies of them have been hampered by the difficulty encountered in expressing them recombinantly in bacterial hosts. We describe here methods for their efficient production in baculovirus-infected insect cells.

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 ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

全球合成的RNA在单细胞水平以下的低氧分析

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

siRNA的筛选识别泛素和泛素样系统压力表生物学途径在培养的哺乳动物细胞

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

Measurement of Heme Synthesis Levels in Mammalian Cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

中血红素的合成水平在哺乳动物细胞中测量

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...