Name: comparison of tobacco host cell protein removal methods by blanching intact plants or by heat treatment of extracts
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Description: Watch this Scientific Journal Video about Comparison of Tobacco Host Cell Protein Removal Methods by Blanching Intact Plants or by Heat Treatment of Extracts at JoVE.com

We would like to acknowledge Dr. Thomas Rademacher, Alexander Boes and Veronique Bei&#223; for providing the transgenic tobacco seeds, and Ibrahim Al Amedi for cultivating the tobacco plants. The authors wish to thank Dr. Richard M. Twyman for editorial assistance as well as G&#252;ven Edg&#252; for providing the MSP1-19 reference. This work was funded in part by the European Research Council Advanced Grant ''Future-Pharma'', proposal number 269110, the Fraunhofer-Zukunftsstiftung (Fraunhofer Future Foundation) and Fraunhofer-Gesellschaft Internal Programs under Grant No. Attract 125-600164.

1. 담배 식물 육성 <ol><li> 0.1 % [w / V] 비료 용액을 1 L로 1 탈 이온수 2 L이어서 각 미네랄 울 블록 플러시. 하나 담배 각 미네랄 울 블록의 씨앗과 씨앗 (16)을 멀리 세척하지 않고 비료 용액 0.25 L와 부드럽게 높이를 놓습니다. </li><li> 70 %의 상대 습도와 온실에서 16 시간의 광주 칠주에 대한 담배 식물 육성 (- 1m - 180 μmol 초 2, λ = 400-700 nm의)과 22분의 25 ℃로 빛 ...

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상기 열 강수량의 세 가지 방법을 효과적으로 모든 크로마토 그래피 정제 단계 (16, 17) 이전에 담배 HCP가 제거 할 수 있습니다. 그들은 예를 들어, 초기 제품 순도, guttation (29), 분비 단백질로 제한됩니다 모두 rhizosecretion 30 원심 추출 (31, 32)을, 증가하는 것을 목표로 다른 전략을 보완합니다. 표적 단백질은 1 개 이상의 분 ~ 60 ° C의 최소의 석출 온도를 ?...

현대 의료 시스템은 점점 바이오 단백질 (1)에 따라 달라집니다. 식물에서 이들 단백질을 생산하는 통상의 발현 시스템 2-4에 비해 낮기 때문에 병원체 부담 더 확장하는 것이 유리하다. 파괴적 추출 절차 탁도 5,000 혼탁 탁도 유닛 (NTUs)를 초과하고, 숙주 세포 단백질 (HCP)의 농도는 종종 95을 초과하는, 높은 입자 부담을 초래하기 때문에, 식물 유래 의약품의 하류 처리 (DSP)가 어려울 수 %의 [m / m] 5,6. 정교한 정화 절차 분산 입자 7-9를 제거하기 위해 필요하지만 크로마토 그래피 장비 HCP에 10,11의 효율적인 제거에 대한 이전 단계가있는 경우 초기 생성물 회수 중에 바인드 - 및 - 용출 모드로 동작하도록 저렴된다. 이것은 floccul를 이용하여 표적 단백질을 침전에 의해 하나를 달성 할 수있다개미 12 낮은 pH 13, 14,뿐만 아니라 HCP가 집계하도록하여. 불로 오스 -1,5- 비스 포스페이트 카르 복실 / 시게나 (RUBISCO), 담배 (된 담배)으로 녹색 식물에서 가장 풍부한 HCP의 선택적 응집은 폴리에틸렌 글리콜 (15)을 첨가하여 촉진하지만, 이것은 고가이고 대형과 양립 할 수있다 -scale 제조. 열처리는 예컨대 Vax8 같은 단백질 말라리아 백신 후보 솔루션 16-18에서 안정적으로 유지하면서, 변성 및 담배 HCP가 95 % 이상을 침전하는 것으로 나타났다. 온도 조절은 혈관을 교반 (II) (I) 희게, 즉 고온의 액체는 그대로 잎의 침지, 및 (iii) 열교환 기 (세 가지 접근법은 담배 HCP에의 열 유도 된 침전을 달성하기 위해 사용 된 그림 1) 16. 그대로 잎의 경우, HCP가의 신속하고 효율적인 침전을 달성하고도 쉬웠다 희게확장 및 식물 바이오 매스 (19)를 세척하는 초기 단계를 포함 기존의 대규모 제조 공정과 호환합니다. 대조적으로, 온도 조절 용기 이미 일부 공정에 사용할 수 있으며 탱크의 표면 대 부피 비율이 점차 감소되기 때문에 식물의 열처리 (20)를 추출하지만, 그 확장 성 및 에너지 전송 속도가 제한되어 사용될 수 있으며 프로세스 규모에 적합하게된다. 열교환 교반 용기를 가열 할 수있는 기술적으로 잘 정의 된 대안이지만 가열 및 냉각 매체, 예를 들면 증기와 냉수의 풍부한 공급뿐만 아니라, 열 ​​교환기 형상에 적응되는 엄격하게 제어 체적 유량을 필요 매체 특성, 예를 들면., 비열. 이 문서는 세 가지 방법이 일반적으로 담배 HCP가, 식물 HCP가의 가열에 의한 침전에 사용할 수있는 방법을 보여줍니다. 설립 및 EAC의 동작실험실 환경에서 H 방식은 대규모 프로세스의 적합성을 평가하기 위해 사용될 수있다. 주요 과제는 공정 규모의 제조시 사용되는 장치 및 조건을 닮은 각 작업에 대한 적절한 스케일 다운 모델과 실행 조건을 확인하는 것입니다. 여기에 제시된 데이터는 DsRed 16 말라리아 백신 후보 Vax8 형광 단백질을 발현하는 형질 전환 담배 식물 실시한 실험을 참조하지만,이 방법은 성공적 N.에 적용된 benthamiana 식물 일시적 다른 바이오 21 단백질 발현. 디자인의-실험 (DOE는) 공정 개발을 용이하게 할 수있다 (22)에 접근, 이전 8 설명한 바와 같이 (23)는이 상황에서 도움이 될 수 있습니다 응집제. 창백 가열 용기 및 열교환 기의 주요 차이점은 창백 구약 반면 프로세스 초기에 그대로 잎에 적용된다그녀는 식물 추출물 (그림 1)에 적용됩니다. <img alt="그림 1" src="/files/ftp_upload/54343/54343fig1.jpg" /> 그림 1 :. 담배 HCP 열 강수량에 대한 세 가지 다른 방법의 구현을 설명 프로세스 흐름 방식은 식물 재료 세척 및 정화 및 정화 전에 균질화한다. 희게 단계 (적색) 용 장비를 쉽게 기존의 장치에 추가 될 수있다. 대조적으로, 교반 용기 (오렌지) 특히 열교환 기 (파란색)를 사용하여 하나 또는 여러 개의 추가 장치 및 배관을 필요로한다. <a href="https://www.jove.com/files/ftp_upload/54343/54343fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a>

<table border="0" cellpadding="0" cellspacing="0" width="1026">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">2100P Portable Turbidimeter</td>
			<td style="width:195px;">Hach</td>
			<td style="width:119px;">4650000</td>
			<td style="width:352px;">Turbidimeter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Amine Coupling Kit</td>
			<td style="width:195px;">GE Healthcare</td>
			<td style="width:119px;">BR100050&#160;</td>
			<td>SPR chip coupling kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Autoclaving basket</td>
			<td style="width:195px;">Nalgene</td>
			<td style="width:119px;">6917-0230</td>
			<td>Basket for leaf blanching</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Biacore T200</td>
			<td style="width:195px;">GE Healthcare</td>
			<td style="width:119px;">28-9750-01</td>
			<td>SPR device</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Bio Cell Analyser BCA 003 R&#38;D with 3D ORM</td>
			<td style="width:195px;">Sequip</td>
			<td style="width:119px;">n.a.</td>
			<td>Particle size analyzer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Blender</td>
			<td style="width:195px;">Waring</td>
			<td style="width:119px;">800EG</td>
			<td>Blender</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">BP-410</td>
			<td style="width:195px;">Furh</td>
			<td style="width:119px;">2632410001</td>
			<td>Bag filter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Centrifuge 5415D</td>
			<td style="width:195px;">Eppendorf</td>
			<td style="width:119px;">5424 000.410</td>
			<td>Centrifuge</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Centrifuge tube 15 mL</td>
			<td style="width:195px;">Labomedic</td>
			<td style="width:119px;">2017106</td>
			<td>Reaction tube</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Centrifuge tube 50 mL self-standing</td>
			<td style="width:195px;">Labomedic</td>
			<td style="width:119px;">1110504</td>
			<td>Reaction tube</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">CM5 chip</td>
			<td style="width:195px;">GE Healthcare</td>
			<td style="width:119px;">BR100012&#160;</td>
			<td>Chip for SPR measurements</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Cuvette 10x10x45</td>
			<td style="width:195px;">Sarsted</td>
			<td style="width:119px;">67.754</td>
			<td>Cuvette for Zetasizer Nano ZS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Design-Expert(R) 8</td>
			<td style="width:195px;">Stat-Ease, Inc.</td>
			<td style="width:119px;">n.a.</td>
			<td>DoE software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Disodium phosphate</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">&#160;4984.3&#160;</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Ferty 2 Mega</td>
			<td style="width:195px;">Kammlott</td>
			<td style="width:119px;">5.220072</td>
			<td>Fertilizer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Forma -86C ULT freezer</td>
			<td style="width:195px;">ThermoFisher</td>
			<td style="width:119px;">88400</td>
			<td>Freezer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Greenhouse</td>
			<td style="width:195px;">n.a.</td>
			<td style="width:119px;">n.a.</td>
			<td>For plant cultivation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Grodan Rockwool Cubes 10x10cm</td>
			<td style="width:195px;">Grodan</td>
			<td style="width:119px;">102446</td>
			<td>Rockwool block</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Twentey-loop heat exchanger (4.8 m length)</td>
			<td style="width:195px;">n.a. (custom design)</td>
			<td style="width:119px;">n.a.</td>
			<td>Heat exchanger</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">HEPES</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">9105.3</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">K200P 60D</td>
			<td style="width:195px;">Pall</td>
			<td style="width:119px;">5302303</td>
			<td>Depth filter layer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">KS50P 60D</td>
			<td style="width:195px;">Pall</td>
			<td style="width:119px;">B12486</td>
			<td>Depth filter layer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Lauda E300</td>
			<td style="width:195px;">Lauda Dr Wobser GmbH</td>
			<td style="width:119px;">Z90010</td>
			<td>Water bath thermostat</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">L/S 24</td>
			<td style="width:195px;">Masterflex</td>
			<td style="width:119px;">SN-06508-24</td>
			<td>Tubing</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:360px;">mAb 5.2</td>
			<td style="width:195px;">American Type Culture Collection</td>
			<td style="width:119px;">HB-9148</td>
			<td>Vax8 specific antibody</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Masterflex L/S</td>
			<td style="width:195px;">Masterflex</td>
			<td style="width:119px;">HV-77921-75</td>
			<td>Peristaltic pump</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Miracloth</td>
			<td style="width:195px;">Labomedic</td>
			<td style="width:119px;">475855-1R</td>
			<td>Filter cloth</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">MultiLine Multi 3410 IDS</td>
			<td style="width:195px;">WTW</td>
			<td style="width:119px;">WTW_2020</td>
			<td>pH meter / conductivity meter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Osram cool white 36 W</td>
			<td style="width:195px;">Osram</td>
			<td style="width:119px;">4930440</td>
			<td>Light source</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Phytotron</td>
			<td style="width:195px;">Ilka Zell</td>
			<td style="width:119px;">n.a.</td>
			<td>For plant cultivation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Sodium disulfit</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">8554.1</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Sodium chloride</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">P029.2</td>
			<td>Media component</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:360px;">Stainless-steel vessel; 0.7-kg 2.0-L; height 180 mm; diameter 120 mm</td>
			<td style="width:195px;">n.a. (custom design)</td>
			<td style="width:119px;">n.a.</td>
			<td>Container for heat precipitation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Synergy HT</td>
			<td style="width:195px;">BioTek</td>
			<td style="width:119px;">SIAFRT</td>
			<td>Fluorescence and spectrometric plate reader</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">VelaPad 60</td>
			<td style="width:195px;">Pall</td>
			<td style="width:119px;">VP60G03KNH4</td>
			<td>Filter housing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Zetasizer Nano ZS</td>
			<td style="width:195px;">Malvern</td>
			<td style="width:119px;">ZEN3600</td>
			<td>DLS particle size distribution measurement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Zetasizer Software v7.11</td>
			<td style="width:195px;">Malvern</td>
			<td style="width:119px;">n.a.</td>
			<td>Software to operate the Zetasizer Nano ZS device</td>
		</tr>
	</tbody>
</table>

comparison of tobacco host cell protein removal methods by blanching intact plants or by heat treatment of extracts

식물은 인간 식품, 사료 원료를 제공 할뿐만 아니라, 항체, 백신 후보로서 바이오 효소 단백질에 대한 경제적 인 생산 시스템으로 개발되었다 아닙니다. 이들은 식물 바이오 매스로부터 정제해야하지만, 크로마토 그래피 단계는 식물 추출물의 숙주 세포 단백질 (HCP가)의 높은 농도에 의해 방해된다. 그러나, 대부분의 HCP가 비가 역적 목적 단백질의 정제를 용이 이후 60 ° C 이상의 온도에서 응집. 여기에, 세 가지 방법은 그대로 잎 또는 추출물 중 하나에서 담배 HCP가의 열 침전을 달성하기 위해 제공됩니다. 그대로 잎의 창백 쉽게 기존 프로세스에 통합 될 수 있지만, 이후 여과 단계에 부정적인 영향을 미칠 수있다. 반대의 경우와 같은 처리 장치의 설계에 큰 변화 불구 하류 작업의 성능을 향상시킬 수있는 교반 용기 잎 추출물의 열 침전 사실이다균질 형상. 마지막으로, 열교환 기 설치 잘 열전달 조건의 관점에서 특성화 스케일 쉽지만 세정 어려울 수 있고, 필터의 용량에 부정적인 영향이있을 수있다. 디자인 수준의 실험 방법을 HCP 제거 및 생성물 회수에 영향을 미치는 가장 중요한 공정 매개 변수를 식별하는데 사용될 수있다. 이것은 다른 식 플랫폼의 각 방법 및 주어진 정제 전략을위한 최적의 방법의 식별의 적용을 용이하게한다.

치면 담배 숙주 세포 단백질의 열 침전 제 2 항에 기재된 희게 절차가 성공적으로 담배는 96 ± 1 %까지 TSP를 감소 70 ° C로 나뭇잎 HCP가 석출 하였다 (N = 3) 따라서, Vax8 표적 단백질의 51 %까지 회복은 증가하면서 크로마토 그래피 분리 16 이전에 0.1 % ~ 1.2에서 순도. 이 64.1 %로 3.3 %에서 순도를 증가 형광 단백질을 DsRed 83 ± 1 % (N = 3)를 복구하는 것이 가능했다...

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Comparison of Tobacco Host Cell Protein Removal Methods by Blanching Intact Plants or by Heat Treatment of Extracts

효과적으로 담배에서 숙주 세포 단백질 (HCP가)의 90 % 이상을 제거 침전 방법 나타난 세 개의 열은 다른 정제 단계 이전에 추출한다. 공장 HCP가이 비가 역적으로 60 ° C 이상의 온도에서 집계.

본래 식물을 표백제로 또는 추출물의 열처리에 의한 담배 숙주 세포 단백질 제거 방법의 비교

Plants not only provide food, feed and raw materials for humans, but have also been developed as an economical production system for biopharmaceutical ...

Research

JoVE Journal

Biology

Preparation and Fractionation of Xenopus laevis Egg Extracts

Xenopus laevis 에그의 작성과 분류는 압축을 풉니다

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical ...

연구

생물학

Detection of Post-translational Modifications on Native Intact Nucleosomes by ELISA

엘리사에 의해 네이티브 손상 Nucleosomes에 포스트 translational 수정의 감지

The genome of eukaryotes exists as chromatin which contains both DNA and proteins. The fundamental unit of chromatin is the nucleosome, which contains 146 ...

The MultiBac Protein Complex Production Platform at the EMBL

EMBL에서 MultiBac 단백질 복잡한 생산 플랫폼

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

Protein-protein Interactions Visualized by Bimolecular Fluorescence Complementation in Tobacco Protoplasts and Leaves

담배 원형질체 및 잎에 이분자 형광 보완에 의해 시각 단백질 - 단백질 상호 작용

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

성인 마우스 두개골 골수에서 조혈 줄기 및 전구 세포의 생체 내 4 차원 추적에서

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

대변​​ 글루코 코르티코이드 분석 : equids는 비 침습적 부신 모니터링

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

DamID-seq: Genome-wide Mapping of Protein-DNA Interactions by High Throughput Sequencing of Adenine-methylated DNA Fragments

DamID-SEQ : 아데닌 - 메틸화 된 DNA 단편의 높은 처리량 시퀀싱에 의해 단백질 DNA 상호 작용의 게놈 넓은 매핑

The DNA adenine methyltransferase identification (DamID) assay is a powerful method to detect protein-DNA interactions both locally and genome-wide. It is ...

Procedure to Evaluate the Efficiency of Flocculants for the Removal of Dispersed Particles from Plant Extracts

절차 식물 추출물에서 분산 된 입자의 제거를위한 응집제의 효율성을 평가하는

Plants are important to humans not only because they provide commodities such as food, feed and raw materials, but increasingly because they can be used ...

Comparison of Three Different Methods for Determining Cell Proliferation in Breast Cancer Cell Lines

유방암 세포주에서 세포 증식을 결정하기위한 세 가지 방법 비교

Measuring cell proliferation can be performed by a number of different methods, each with varying levels of sensitivity, reproducibility and compatibility ...

Xenopus Oocytes: Optimized Methods for Microinjection, Removal of Follicular Cell Layers, and Fast Solution Changes in Electrophysiological Experiments

Xenopus Oocytes: Optimized Methods for Microinjection, Removal of Follicular Cell Layers, and Fast Solution Changes in Electrophysiological Experiments

Xenopus의 난 모세포 : 미세 주입을위한 최적화 방법, 여포 세포 레이어의 제거 및 전기 생리 실험에서 빠른 해결 방법 변경

The Xenopus oocyte as a heterologous expression system for proteins, was first described by Gurdon et al.1 and has been widely used since its discovery ...