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

우리는 최대한의 효율과 수율에 대한 프로토콜을 최적화 도움이 로스 랩의 사전 구성원을 인정하고 싶습니다.

인플루엔자 H3N2 VLP의 생성 1.​​ 포유 동물 발현 시스템 <ol><li> 이러한 서브 클론 전술 pTR600 진핵 발현 벡터 내로 구조적 바이러스 당 단백질 헤 마글 루티 닌 (HA), 뉴 라미니다 제 (NA), 및 인간 면역 결핍 바이러스 (HIV) 개그 P55. 7 </li><li> 화학적 능력 대장균 (예 DH5α)에서 DNA를 증폭하고 제조자의 지시에 따라 플라스미드 정제 키트를 사용하여 형질 급 플라스미드를 분리. DNA의 양 수...

<ol>
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우리는 성공적으로 SVPs 다양한 병원균에 대해 여러 구조 단백질로 구성 VLP를 표현하고 정화 위에서 설명한 기술을 사용했다. 일반적으로, 우리는 VLPs를 생성하는 포유류 발현 시스템을 사용한다. 그러나, 우리의 손에, 포유 동물 세포는 CHIK의 VLP 수익률이 낮았다 산출했다. 재조합 배큘로 바이러스 곤충 세포 시스템을 사용하는 경우 CHIK VLP의 수율은보다 강력했다. 일반적으로, 배큘로 바이러스 곤...

바이러스 유사 입자 (VLP를)은 인간의 예방 접종에 대한 승인 된 기술입니다. 실제로, 인간 유두종 바이러스 (HPV) 및 간염 Β 포함한 더 현대적 허가 백신의 일부 (HepΒ) 백신이 방법을 사용한다. VLP를이 자기 ​​조립이 가능한 구조 단백질로부터 형성된다. 조립 된 입자는 바이러스 모폴로지을 모방하지만, 감염 또는 바이러스 성 게놈이 부족하기 때문에 복제 할 수 없습니다. VLP를가 표현 원핵 생물과 진핵 생물 시스템의 수와 정제 할 수있다. - 28 %, 효모 - 20 %, 식물 - 9 %, 곤충 - 28 %, 그리고 포유류 - 15 % 1 박테리아 : 문헌의 검토는 서로 다른 발현 시스템은 다음과 같은 비율로 사용되는 것으로 나타났습니다. 참고로, L1 캡시드 단백질을 기반으로 HPV 백신은 효모 (가다실) 또는 곤충 전지 시스템 (서바릭스)이 생산됩니다. HepΒ 백신, 및 Recombivax Engerix-Β 또한 효모에서 생산 한 HepΒ 표면 항원 (3)로 구성되는4. 우리는 여러 조립 구조 단백질의 공동 발현을 요구하는 VLPs를 생성하는 포유 동물 및 곤충 세포 발현 시스템을 사용한다. 인플루엔자 바이러스, 호흡기 세포 융합 바이러스 (RSV), 뎅기열 바이러스 (DENV), 및 chikungunya 바이러스 (CHIKV) : 우리의 작업은 인간의 병원균에 대한 설계, 생산 및 정화 VLP 기반 백신에 초점을 맞추고 있습니다. 우리의 방법은 다수의 발현 플라스미드의 다중 구조 단백질의 공동 발현 또는 단일 발현 플라스미드 (도 1)을 허용 할 정도로 유연하다. 이전에는 제조 및 정제 H5N1 VLPs를 인간 배아 신장 293T 세포 5,6-에서 인플루엔자 헤 마글 루티 닌 (HA), 뉴 라미니다 제 (NA), 매트릭스 (M1)을 코딩하는 플라스미드의 공동 발현 조립. 유전자는 포유 동물 세포에서의 발현을 위해 코돈 최적화 pTR600, transc을 개시 사이토 메갈로 바이러스의 즉시 초기 프로모터 플러스 인트론을 포함하는 진핵 세포 발현 벡터에 클로닝진핵 삽입의 ription 및 전사 (7)의 종료에 대한 소 성장 호르몬 폴리아 데 닐화 신호. HA, NA (도 1A) 및 다른 바이러스의 매트릭스 단백질, HIV 개그 P55,의 3 플라스미드의 공동 발현을 사용하여 유사한 접근 방식이 연구에서 인간 계절 인플루엔자 아형 H3N2 VLPs를 생성을 위해 사용하고, 생성하는 것으로 나타났다 인플루엔자 입자 (8)과 비슷한 크기의 VLP를. 인플루엔자 백신은 주로 항 HA 항체를 유도하지만, 인플루엔자 뉴 라미니다 제를 첨가하는 형질 세포 (9)와 같은 본 추가 면역 원성 표적에서 신진 VLP 있도록 시알 리다 아제 활성을 매개한다. RSV VLP를 생산하기 위해, 우리는 또한 이전에 기술과 전자 현미경 6,10 특징으로 추가, HIV 개그를 사용하여 VLP 형성의 유연성을 보여주기 위해 독점적 RSV 표면 당 단백질을 제시 원형 백신을 설계하는 HIV 개그의 관련이없는 코어를 선택했다. 기타이전 즉 RSV 당 단백질 뉴캐슬 질병 바이러스 (NDV) (11), 및 인플루엔자 매트릭스 (12)로부터 각종 바이러스 요소를 사용하여 조립 될 수 제시 VLPs를 보여 주었다. 전체 길이 표면 당 단백질 서열은 효소 면역 분석법 (ELISA)을 통해 기능성 수용체 결합 및 항체 인식의 분석에 대한 필요가있을 수있다 천연 배좌를 유지 본 연구에 이용 하였다. 입자를 생성하는 하나의 플라스미드 발현 시스템의 사용에 대한 우리의 예는 DENV 및 CHIKV 있습니다. DENV의 경우에, 우리는 PRM / E 구조 유전자 발현 카세트 (13)를 함유 한 플라스미드를 293T 세포 없음 캡시드와 서브 바이러스 레 입자 (SVPs)를 생산할 수있다. 용어 SVP가 바이러스 구조 단백질의 조립에서 코어 또는 캡시드 단백질의 부재를 나타 내기 위해 사용된다. CHIK VLP를도 CHIKV 구조 유전자 카세트 부호화 캡시드 및 외피 단백질을 함유 한 플라스미드를 사용하여 제조 될 수 있고, 또는 I의재조합 배큘로 바이러스 곤충 세포 발현 (- C도 1b)을 위해 최적화 동일한 구조 유전자 카세트를 인코딩 감염시켜 세포 nsect. 상술 된 접근법 식의 최종 결과는 20 % 글리세롤 쿠션을 초 원심 분리를 통해 정제 할 수있는 세포 배양 배지에 VLP를 릴리스한다. 이 보고서에서 우리는 표현과 포유 동물 및 곤충 세포 시스템에서 이러한 VLP를 정화하는 방법을 제시한다.

<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

바이러스 유사 입자 (VLP를) 및 서브 바이러스 레 입자 (SVPs)은 살아있는 병원체의 사용을 통해 증대 바이오 안정성의 이점을 제공하는 바이러스 백신 디자인에 대한 대체 방법이다. 비 감염성 및자가 조립 VLPs를 살아있는 병원체 또는 항원 전달을위한 재조합 바이러스 벡터의 필요성을 우회 면역원으로 구조 단백질을 제공하는 데 사용된다. 이 글에서, 우리는 전임상 동물 실험에서 미래의 애플리케이션을위한 VLP 설계 및 개발의 여러 단계를 보여줍니다. 절차는 다음 단계를 포함 항원 투여에 대한 항원의 선택, 선택의 세포주, VLP를 / SVPs의 정제 항원의 발현 및 정량화. 우리는 우리의 항원의 발현 모두 포유 동물 및 곤충 세포 라인의 사용을 설명하고 방법은 수율이 다른 방법을 보여줍니다. 제시된 방법은 병원체의 종류에 적용 할 수있는 면역 원성 항원 STR로 대체함으로써 달성 될 수있다관심 사용자 미생물 uctural 단백질. VLP를하고 SVPs는 항원의 특성과 최고의 백신 후보의 선택에 도움이됩니다.

VLP 수익률은 바이러스 항원 구조 설계에 의한 변수했다. 이 프로토콜에서는, 우리는 초 원심 분리하여 상층 액의 SVP 또는 VLP를 생산 및 정제를위한 곤충과 포유 동물 세포의 사용을 증명하고있다. DENV PRM / E 구조 유전자 발현 카세트의 네 아형 표 1 (1-4으로 경계가) DENV SVPs의 버전을 구성하고 0.6 ml의 양으로 총 단백질 mg을 1.1-2.6의 범위를 보여주기 위해 사용되?...

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

여기서는 배큘로 바이러스 또는 포유류 발현 시스템, 및 초 원심 정제를 사용하여 바이러스 입자의 합성을위한 프로토콜을 제시한다. 이것은 매우 최적화 방법은 안전하고 유연하게 타겟으로 백신 바이러스 항원을 확인하는 데 사용된다.

표현과 예방 접종에 대한 바이러스 입자의 정제

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.

Electroporation에 대한 Murine 골수에서 조혈 세포 차 문화의 준비

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

연구

생물학

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.

유기 용매 및 초산없이 쿠매시 G - 250로 젤의 단백질 염색법

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.

고효율 기본 셀을 Transfect하는 진 Pulser Electroporation MXcell의 시스템을 사용하여

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.

유전자 발현 연구를 단순화하는 자동 셀 카운터를 사용하여 : Namalwa 셀에서 IL - 4 종속 유전자 발현의 siRNA의 최저

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.

표현, 세제 Solubilization 및 멤브레인 전송, MexB의 Multidrug 저항 단백질의 정제

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.

의 파라핀 - 임베디드 및 냉동 섹션 Drosophila 성인 근육

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.

활성 T.의 체외의 Reconstitution에서 castaneum의 Telomerase

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.

의 낭성 섬유증의 Transmembrane의 전도성 조절 단백질의 표현과 정제 Saccharomyces cerevisiae

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.

성적 증명서와 Metabolites에서의 정화<em&gt; Drosophila</em&gt; 헤드

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.

저산소증에 따라 단일 세포 수준에서 글로벌 RNA의 합성 분석

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