Name: expression and purification of virus-like particles for vaccination
Uploaded: 2016-06-02T15:00:00
Description: Watch this Scientific Journal Video about Expression and Purification of Virus-like Particles for Vaccination at JoVE.com

我们要感谢罗斯实验室谁帮助优化效率最高和产量协议的前成员。

流感H3N2 VLP的产生1.真核表达系统<ol><li>亚克隆病毒结构的糖蛋白血凝素（HA），神经氨酸酶（NA），和人免疫缺陷病毒（HIV）的Gag的p55到真核表达载体中，如先前所描述pTR600 7 </li><li>扩增的DNA在化学感受大肠杆菌（ 例如 ，DH5α）和使用质粒纯化试剂盒按照制造商的说明分离转染级的质粒。的DNA的量依赖于产率和用户的需要。 </li><li>保持哺乳动物细胞系，293T，在含有10％胎牛血清（...

<ol>
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我们使用以上描述的技术成功表达并纯化的SVP和各种病原体多种结构蛋白组成的VLP。在一般情况下，我们使用哺乳动物表达系统，以产生我们的VLP。然而，在我们的手中，哺乳动物细胞衍生CHIK VLP收益率较低。使用重组杆状病毒昆虫细胞系统时，CHIK VLP产量为更加强劲。在一般情况下，杆状病毒 - 昆虫细胞系统产生的同时控制下更高量的重组蛋白，这可能是由于，并且还由于高水平的外源基因的表...

病毒样颗粒（VLP）是人类疫苗接种核准技术。事实上，一些比较现代感许可的疫苗，包括人乳头瘤病毒（HPV）和肝炎Β的（HepΒ）疫苗采用这种方法。的VLP从能够自组装的结构蛋白形成。组装的粒子模仿病毒形态，但不能感染或复制，因为它们缺乏病毒基因组。的VLP可表达并从许多原核和真核系统的纯化。对文献的审查表明，不同的表达系统按以下标准使用：细菌- 28％，酵母- 20％，植物- 9％，昆虫- 28％，和哺乳动物- 15％1。值得注意的是，基于L1衣壳蛋白HPV疫苗是在酵母中（Gardasil的），或在昆虫细胞系统（的Cervarix）2制备。 HepΒ疫苗，Recombivax和的Engerix-Β，也产生了酵母和组成HepΒ表面抗原3的4。 我们使用哺乳动物和昆虫细胞表达系统来产生需要用于组装多个结构蛋白的共表达的VLP。我们的工作主要集中在设计，生产和净化基于VLP的疫苗对人类病原体：流感病毒，呼吸道合胞病毒（RSV），登革热病毒（DENV）和基孔肯雅病毒（CHIKV）。我们的方法是足够的灵活性，以允许从多个表达质粒的多种结构蛋白的共表达，或单个表达质粒（ 图1）。先前，我们制备并纯化的H5N1的VLP从编码人胚胎肾293T细胞5,6-流感血凝素（HA），神经氨酸酶（NA），和基质（M1）的质粒的共表达装配。用于在哺乳动物细胞中表达的基因进行密码子优化，并克隆到pTR600含有巨细胞病毒立即早期启动子加上内含子的用于启动创见一个真核表达载体真核刀片ription和转录7终止牛生长激素聚腺苷酸化信号。用于产生人类季节性流感亚型H3N2的VLP的本研究中使用的HA，NA（ 图1A）和另一种基质蛋白，HIV的Gag的p55，三个质粒共表达了类似的方法，并已被证明产生大小颗粒流感8种相似的病毒样颗粒。虽然流感疫苗主要诱导抗HA抗体，增加流感病毒神经氨酸酶介导唾液酸酶的活性，使VLP转染细胞的9，也提出了另外的免疫原性目标崭露头角。以产生的RSV的VLP，我们还选择的HIV Gag的的无关核心来设计呈现完全RSV表面糖蛋白，以进一步展示使用HIV Gag的VLP形成灵活性，如先前描述和表征通过电子显微镜6,10原型疫苗。其他先前已经表明，病毒样颗粒呈现呼吸道合胞病毒糖蛋白可以利用新城疫病毒（NDV）11和流感基质12多种病毒组件进行组装。全长表面糖蛋白的序列在本研究中利用保留天然构象可能是必要的功能性受体结合和抗体识别测定通过酶联免疫吸附试验（ELISA）。 我们使用单一质粒表达系统来生成颗粒的例子是DENV和CHIKV。在登革病毒的情况下，我们可以生产亚病毒颗粒（的SVP）从含有的prM / E的结构基因的表达盒13中的单个质粒中的293T细胞无衣壳。术语SVP用于表示在病毒结构蛋白的组装缺少核心或衣壳蛋白。 CHIK的VLP也可使用含有CHIKV结构基因盒，编码衣壳和包膜蛋白的单个质粒产生的，或在我NSECT细胞通过用重组杆状病毒编码相同的结构基因盒为昆虫细胞表达（ 图1B - C）的优化感染。 表达办法上面讨论的最终结果是VLP的释放到可随后通过超速离心通过20％甘油缓冲纯化细胞培养液中。在这份报告中，我们提出的方法来表达和哺乳动物和昆虫细胞系统净化这些病毒样颗粒。

<table border="0" cellpadding="0" cellspacing="0" width="786">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Plasmid Maxi Kit</td>
			<td style="width:167px;">Qiagen</td>
			<td style="width:119px;">12163</td>
			<td style="width:247px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">DMEM</td>
			<td style="width:167px;">Cellgro</td>
			<td>10-013</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Lipofectamine</td>
			<td style="width:167px;">Life Technologies</td>
			<td>L3000075</td>
			<td>Lipofectamine 2000, Lipofectamine 3000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Opti-MEM I</td>
			<td style="width:167px;">Life Technologies</td>
			<td>31985062</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Bac-to-Bac Baculovirus Expression System</td>
			<td style="width:167px;">Life Technologies</td>
			<td style="width:119px;">10359-016</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Cimarec I 6 multipoint stirrer plate</td>
			<td style="width:167px;">ThermoFisher Scientific</td>
			<td style="width:119px;">50094596</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Polyclear ultracentrifuge tubes</td>
			<td style="width:167px;">Seton</td>
			<td style="width:119px;">7025</td>
			<td>Confirm appropriate tubes for ultracentrifuge and bucket size</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Micro BCA Protein Assay Kit</td>
			<td style="width:167px;">ThermoFisher Scientific</td>
			<td style="width:119px;">23235</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Phosphate citrate buffer</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:119px;">P4922</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">o-phenylenediamine dihydrochloride&#160;</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:119px;">P8287</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">0.22 &#956;m vacuum filter top (500 mL)</td>
			<td style="width:167px;">Nalgene</td>
			<td style="width:119px;">569-0020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Glycerol</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:119px;">G5516</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">H2SO4</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:119px;">339741&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Sf-900 II SFM</td>
			<td style="width:167px;">ThermoFisher Scientific</td>
			<td style="width:119px;">10902-096</td>
			<td></td>
		</tr>
	</tbody>
</table>

expression and purification of virus-like particles for vaccination

病毒样颗粒（VLPs）和亚病毒颗粒（的SVP）是另一种方法，以病毒疫苗的设计，在使用活病原体提供了更高的生物安全性和稳定性的优点。非感染和自组装，的VLP被用来呈现结构蛋白作为免疫原，绕过活病原体或用于抗原递送的重组病毒载体的需要。在这篇文章中，我们证明VLP设计和开发的不同阶段的临床前动物试验未来的应用。该过程包括以下阶段：抗原的选择，在选择的细胞系的VLP / SVP的纯化抗原的表达，并量化为抗原剂量。我们演示如何使用哺乳动物和昆虫细胞系，为我们的抗原表达和展示方法产量的差异。提出的方法可以应用于各种病原体，并且可以通过替换与免疫原性str中的抗原来实现感兴趣的用户的微生物的uctural蛋白质。病毒样颗粒和的SVP协助抗原特性和最佳候选疫苗的选择。

VLP产量分别由病毒抗原结构的设计变量。在这个协议中，我们已经证明了在上清液生产SVP或VLP的纯化通过超速离心使用昆虫和哺乳动物细胞的。 DENV的prM / E的结构基因的表达盒的四种亚型被用来构建DENV的SVP（界定为1-4）的版本于表1，并在0.6毫升卷证明1.1-2.6毫克总蛋白的范围内。对于需要三个基因构建病毒样颗粒，我们发现1优化的DNA转染比：1：2的HA：NA：加...

Watch this Scientific Journal Video about Expression and Purification of Virus-like Particles for Vaccination at JoVE.com

Expression and Purification of Virus-like Particles for Vaccination

这里，我们提出用于合成使用任一杆状病毒或哺乳动物表达系统，并超速离心纯化病毒样颗粒的协议。这种高度可定制的方式被用来识别在一个安全的，灵活的方式的病毒抗原作为疫苗的目标。

表达和疫苗病毒样颗粒的纯化

Virus-like particles (VLPs) and subviral particles (SVPs) are an alternative approach to viral vaccine design that offers the advantages of increased ...

The overall goal of this procedure is to purify virus-like particles, or VLPs, expressed and secreted by mammalian or insect-cell expression systems. This method can answer key questions in a vaccinoly's field, such as how to express and purify conformationally relevant viral antigens in a safe and flexible manner. The main advantage of this technique is that it does not require protein precipitation using polyethylene glycol or preparation of multiple density layers to concentrate VLPs.Though this method can provide insight into the identification of viral antigens as vaccine targets, VLPs can also be used as tools for disease diagnosis and serology. Visual demonstration of this method is critical as a glycerol underlay in VLP resuspension steps are difficult to learn because beginners may fail to exercise proper technique. On the day of transvection prepare liposome and DNA solutions per the manufacturer's recommendations.Transvect the DNA cells with a DNA composition of one to one to two of HA to NA to Gag and a total DNA quantity of 40 micrograms per T150 flask. Repeat this step to create nine T150 flasks, for a total volume of 200 milliliters. Next, dilute the DNA and liposome solutions in serum-free transvection media without antibiotics, so that each flask contains a total volume of 24 milliliters.Return the flasks to the incubator and maintain the cells and transvection culture medium until the day a virus-like particle, or VLP, harvest. Transfer the supernatant into 50 milliliter conical tubes to harvest the culture from the cells after 72 to 96 hours post-transvection. Spin the cells down to pellet the cellular debris.Collect the supernatants and filter them through a 0.22 micron pour membrane. Culture previously prepared spodoptera frugiperda, or Sf9 cells, in suspension in spinner flasks, by continuously stirring at 130 rpm on a multi-point stirrer plate system. Maintain culture volumes at no more than half the volume of the spinner flask for proper aeration.Next, express CHIK VLPs by infecting 250 milliliters of Sf9 cells in a spinner flask at a density of two times ten to the six cells per milliliter with previously prepared recombinant baculovirus, and return the cells to a 28 degree Celsius incubator. Use trypan blue exclusion to determine if the cell viability has decreased to 70 to 80%Upon confirmation, transfer the cultures directly from suspension into 50 milliliter conical tubes and spin the cells down. Collect the supernatants and filter them through a 0.22 micron pour membrane before sedimentation.Sterilize six 25 millimeter by 89 millimeter open top ultracentrifuge tubes with 70%ethanol. Then, ensure the ethanol has dried off completely. Load 32 milliliters of supernatants into the clean tubes.Next, carefully underlay supernatants with three milliliters of sterile 20%glycerol and PBS. Underlay the glycerol slowly to avoid disruption of the thin density layer between the glycerol and the VLP solution. Dispensing the glycerol too quickly will cause the glycerol and the VLP solutions to mix, reducing VLP sedimentation.Make sure the tubes are balanced, then start the spin. After four hours, remove the tubes from the centrifuge. Aspirate the supernatant, being careful not to dislodge the pellet from the tube.Resuspend the sedimented VLPs in at least 100 microliters at the bottom of the tubes with sterile PBS by gently and slowly pipetting up and down. Resuspension of the VLP pellet should be performed with gentle, slow repeated aspiration and expulsion of PBS using a pipette. Avoid the creation of bubbles to limit VLP loss.Finally, store the resuspended VLPs. VLP volumes and total protein yield from a conventional BCA assay were obtained from a variety of SVP and VLP constructs. CHIK SVP yields from Sf9 cells range between 0.008 to 0.016 milligrams of total protein per milliliter of supernatant volume.While production through mammalian 293 T-cells yields a ten-fold reduction of protein. This electron micrograph image shows that HA Gag core influenza VLPs are successfully expressed, assembled and purified as VLPs. Well attempting this procedure it's important to remember to transvect and infect only healthy and viable cell cultures as this will effect overall protein expression levels.Following this procedure, other methods like BCA, ELIZA, and HA assays can be performed in order to measure total protein content, specific antigen content and expression of the functional HA.Don't forget that working with an ultracentrifuge can be dangerous and precautions such as balancing the tubes, using only recommended rotors and speeds, and proper supervision of new lab personnel should always be taken while performing this procedure.

expression-and-purification-of-virus-like-particles-for-vaccination

Research

JoVE Journal

Biology

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 ...

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

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 (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

使用基因脉冲MXcell电穿孔系统主细胞的转染效率高

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

使用自动细胞计数器简化基因表达研究：在Namalwa细胞的IL - 4依赖的基因表达的siRNA击倒

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

Expression, Detergent Solubilization, and Purification of a Membrane Transporter, the MexB Multidrug Resistance Protein

Multidrug resistance (MDR), the ability of a cancer cell or pathogen to be resistant to a wide range of structurally and functionally unrelated anti-cancer drugs or antibiotics, is a current serious problem in public health. This multidrug resistance is largely due to energy-dependent drug efflux pumps. The pumps expel anti-cancer drugs or antibiotics into the external medium, lowering their intracellular concentration below a toxic threshold. We are studying multidrug resistance in Pseudomonas aeruginosa, an opportunistic bacterial pathogen that causes infections in patients with many types of injuries or illness, for example, burns or cystic fibrosis, and also in immuno-compromised cancer, dialysis, and transplantation patients. The major MDR efflux pumps in P. aeruginosa are tripartite complexes comprised of an inner membrane proton-drug antiporter (RND), an outer membrane channel (OMF), and a periplasmic linker protein (MFP) 1-8. The RND and OMF proteins are transmembrane proteins. Transmembrane proteins make up more than 30% of all proteins and are 65% of current drug targets. The hydrophobic transmembrane domains make the proteins insoluble in aqueous buffer. Before a transmembrane protein can be purified, it is necessary to find buffer conditions containing a mild detergent that enable the protein to be solubilized as a protein detergent complex (PDC) 9-11. In this example, we use an RND protein, the P. aeruginosa MexB transmembrane transporter, to demonstrate how to express a recombinant form of a transmembrane protein, solubilize it using detergents, and then purify the protein detergent complexes. This general method can be applied to the expression, purification, and solubilization of many other recombinantly expressed membrane proteins. The protein detergent complexes can later be used for biochemical or biophysical characterization including X-ray crystal structure determination or crosslinking studies.

In this protocol we demonstrate the expression, solubilization, and purification of a recombinantly expressed membrane protein, MexB, as a soluble protein detergent complex. MexB is a multidrug resistance membrane transporter from the opportunistic bacterial pathogen Pseudomonas aeruginosa.

表达，洗涤剂溶解，和一个膜转运，MexB多药耐药蛋白的纯化

Multidrug resistance (MDR), the ability of a cancer cell or pathogen to be resistant to a wide range of structurally and functionally unrelated ...

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into muscle physiology. Therefore, identifying and characterizing molecular mechanisms that underlie muscle damage is critical. The structure of adult Drosophila multi-fiber muscles resemble vertebrate striated muscles 1 and the genetic tractability of Drosophila has made it a great system to analyze dystrophic muscle morphology and characterize the processes affecting muscular function in ageing adult flies 2. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. These preparations allow for the tissue to be stained with classical histological stains and labeled with protein detecting dyes, and specifically cryosections are ideal for immunohistochemical detection of proteins in intact muscles. This allows for analysis of muscle tissue structure, identification of morphological defects, and detection of the expression pattern for muscle/neuron-specific proteins in Drosophila adult muscles. These techniques can also be slightly modified for sectioning of other body parts.

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Identification of mechanisms underlying muscle damage is crucial. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. This allows analysis of muscle morphology and localization of protein and other muscle cell components.

石蜡包埋及冷冻的部分果蝇成人肌肉

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

在体外重组活跃T。谷盗端粒酶

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel, that when mutated, can give rise to cystic fibrosis in humans.There is therefore considerable interest in this protein, but efforts to study its structure and activity have been hampered by the difficulty of expressing and purifying sufficient amounts of the protein1-3. Like many 'difficult' eukaryotic membrane proteins, expression in a fast-growing organism is desirable, but challenging, and in the yeast S. cerevisiae, so far low amounts were obtained and rapid degradation of the recombinant protein was observed 4-9. Proteins involved in the processing of recombinant CFTR in yeast have been described6-9 .In this report we describe a methodology for expression of CFTR in yeast and its purification in significant amounts. The protocol describes how the earlier proteolysis problems can be overcome and how expression levels of CFTR can be greatly improved by modifying the cell growth conditions and by controlling the induction conditions, in particular the time period prior to cell harvesting. The reagants associated with this protocol (murine CFTR-expressing yeast cells or yeast plasmids) will be distributed via the US Cystic Fibrosis Foundation, which has sponsored the research. An article describing the design and synthesis of the CFTR construct employed in this report will be published separately (Urbatsch, I.; Thibodeau, P. et al., unpublished). In this article we will explain our method beginning with the transformation of the yeast cells with the CFTR construct - containing yeast plasmid (Fig. 1). The construct has a green fluorescent protein (GFP) sequence fused to CFTR at its C-terminus and follows the system developed by Drew et al. (2008)10. The GFP allows the expression and purification of CFTR to be followed relatively easily. The JoVE visualized protocol finishes after the preparation of microsomes from the yeast cells, although we include some suggestions for purification of the protein from the microsomes. Readers may wish to add their own modifications to the microsome purification procedure, dependent on the final experiments to be carried out with the protein and the local equipment available to them. The yeast-expressed CFTR protein can be partially purified using metal ion affinity chromatography, using an intrinsic polyhistidine purification tag. Subsequent size-exclusion chromatography yields a protein that appears to be &gt;90% pure, as judged by SDS-PAGE and Coomassie-staining of the gel.

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

Attempts to express the cystic fibrosis transmembrane conductance regulator (CFTR) in Saccharomyces cerevisiae have, until now, yielded relatively low amounts of protein. This protocol and the associated reagents distributed via the Cystic Fibrosis Foundation should allow the preparation of milligram amounts of this 'difficult' eukaryotic membrane protein.

囊性纤维化跨膜传导调节蛋白表达和纯化酿酒酵母

The  cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride  channel, that when mutated, can give rise to cystic fibrosis in  ...

Purification of Transcripts and Metabolites from Drosophila Heads

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. Although fruit flies provide obvious experimental advantages, research on neurodegenerative diseases has mostly relied on traditional techniques, including genetic interaction, histology, immunofluorescence, and protein biochemistry. These techniques are effective for mechanistic, hypothesis-driven studies, which lead to a detailed understanding of the role of single genes in well-defined biological problems. However, neurodegenerative diseases are highly complex and affect multiple cellular organelles and processes over time. The advent of new technologies and the omics age provides a unique opportunity to understand the global cellular perturbations underlying complex diseases. Flexible model organisms such as Drosophila are ideal for adapting these new technologies because of their strong annotation and high tractability. One challenge with these small animals, though, is the purification of enough informational molecules (DNA, mRNA, protein, metabolites) from highly relevant tissues such as fly brains. Other challenges consist of collecting large numbers of flies for experimental replicates (critical for statistical robustness) and developing consistent procedures for the purification of high-quality biological material. Here, we describe the procedures for collecting thousands of fly heads and the extraction of transcripts and metabolites to understand how global changes in gene expression and metabolism contribute to neurodegenerative diseases. These procedures are easily scalable and can be applied to the study of proteomic and epigenomic contributions to disease.

Purification of Transcripts and Metabolites from Drosophila Heads

We describe here the procedures for the extraction and purification of mRNA and metabolites from Drosophila heads. We are applying these techniques to better understand the cellular perturbations underlying neuronal degeneration. These methodologies can be easily scaled and adapted for other "omic" projects.

成绩单和代谢产物的分离纯化<em&gt;果蝇</em&gt;头

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. ...

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 ...