이 작품은 HA, FCA, 그리고 JDC에 온타리오 에이즈 치료 네트워크 연구 운영 그랜트 (로저-G645) 및 보건 연구 새로운 탐정 수상 (MSH-113554)의 캐나다 연구소 JEL까지, 토론토 장학금의 대학에 의해 지원되었다. 저자 제공 세포, ebolavirus GP의 발현 벡터 및 일반 조언 스크립스 연구소 (라 Jolla, CA)에서 마니 Fusco, Dafna 아벨 박사 에리카 Ollmann Saphire 감사드립니다.

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관심의 어떠한 충돌 선언 없습니다.

10 층 전지 공장 단백질 밀리그램 양의 생산을위한 효과적인 선박입니다. 이러한 롤러 병, 흔들 flasks 또는 회전자 flasks 같은 다른 전통적인 혈관, 이상 세포 공장 사용의 가장 큰 장점은 추가적인 실험실 장비의 구입을 요구하지 않는다는 것입니다. 표준 CO 2 배양기는 (~ 6.0 세제곱. 피트) 쉽게 사 10 층 전지 공장 (그림 2)를 수용합니다. 또한, 이러한 혈관 요리 flasks 또는 세포와 ...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 댓글 </td></tr><tr><td> 알칼리 인산 분해 효소 (BCIP / NBT) 액체 기판 솔루션 </td><td> 시그마 </td><td> B6404 </td><td></td></tr><tr><td> Antimycotic / 항생제, 100X </td><td> Invitrogen </td><td> 15240062 </td><td></td></tr><tr><td> 안티 HA 친화력 매트릭스, 클론 3F10 </td><td> 로슈 </td><td> 1,815,016 </td><td></td></tr><tr><td> 안티 HA murine mAb, 클론 16B12 </td><td> Covance </td><td> MMS-101P </td><td></td></tr><tr><td> 세포 배양 플라스크는 T75 cm이 조직 문화는 치료 </td><td> 코닝 </td><td> 430,641 </td><td></td></tr><tr><td> 세포 배양 플라스크는 T225 cm이 조직 문화는 치료 </td><td> 코닝 </td><td> 431,082 </td><td></td></tr><tr><td>세포 배양 접시는, 6 잘 조직 문화 처리 </td><td> 코닝 </td><td> 3516 </td><td></td></tr><tr><td> 세포 공장, 10 층 CellSTACK </td><td> 코닝 </td><td> 3312 </td><td></td></tr><tr><td> Centramate 오메가 5K 카세트 </td><td> 물리다 </td><td> OS005C12 </td><td></td></tr><tr><td> Centramate 오메가 30K 카세트 </td><td> 물리다 </td><td> OS030C12 </td><td></td></tr><tr><td> 크로마 토그래피 유리 칼럼, 1.0x10 cm </td><td> Kontes </td><td> 4204001010 </td><td></td></tr><tr><td> * 시프 로플 록 사신 </td><td> 시그마 </td><td> 17,850 </td><td></td></tr><tr><td> CO 2 </td><td></td><td></td><td></td></tr><tr><td> Dulbecco의 수정된 독수리의 미디어 (DMEM) </td><td> 시그마 </td><td> D5796 </td><td></td></tr><tr><td> 태아 소 혈청 (FBS), inactivated 열 </td><td> Invitrogen </td><td> 일2484-028 </td><td></td></tr><tr><td> FuGENE의 HD transfection 시약 </td><td> Promega </td><td> 4709691001 </td><td></td></tr><tr><td> GeneJuice의 transfection 시약 </td><td> EMD / 머크 </td><td> 70967-6 </td><td></td></tr><tr><td> 글리신 </td><td> 시그마 </td><td> G8898 </td><td></td></tr><tr><td> 알칼리 염소 안티 - 마우스 IgG F (AB &#39;) 2 </td><td> 써모 과학 </td><td> 31,324 </td><td></td></tr><tr><td> 인산 가수 분해 효소 - 복합 항체 </td><td></td><td></td><td></td></tr><tr><td> 적혈구응집소 (HA) 펩타이드, 100 밀리그램 </td><td> Genscript </td><td> 맞춤형 합성 </td><td></td></tr><tr><td> (순서 : YPYDVPDYA, 95 % 순도) </td><td></td><td></td><td></td></tr><tr><td> HEK 293T 세포 </td><td> ATCC </td><td> CRL-11268 </td><td></td></tr><tr><td> HouseholD 표백제 (4 % W / V 나트륨 차아 염소 산염) </td><td> 다양한 브랜드가 있습니다 </td><td></td><td></td></tr><tr><td> Immobilon-P PVDF 멤브레인 </td><td> Millipore </td><td> IPVH07850 </td><td></td></tr><tr><td> MiniPrep 플라스미드 정제 키트, PureLink 빠른 </td><td> Invitrogen </td><td> K2100-11 </td><td></td></tr><tr><td> MaxiPrep 플라스미드 정제 키트, PureLink HiPure </td><td> Invitrogen </td><td> K2100-07 </td><td></td></tr><tr><td> 난이 3 </td><td> 시그마 </td><td> S8032 </td><td></td></tr><tr><td> pDISPLAY의 발현 벡터 </td><td> Invitrogen </td><td> V660-20 </td><td></td></tr><tr><td> 100X 페니실린은 / 스트렙토 마이신 (펜 / strep) </td><td> Invitrogen </td><td> 15140-122 </td><td></td></tr><tr><td> 인산 버퍼 식염수 (PBS), 무균 1X </td><td> 시그마 </td><td> D8537 </td><td></td></tr><tr><td> Polyethyleneimine (PEI), 선형 25 kDa </td><td> Polyscience </td><td> 23,966 </td><td></td></tr><tr><td> 우유 건조 분말 탈지 </td><td> 카네이션 </td><td></td><td></td></tr><tr><td> Stericup-GP PES 진공 여과 장치, </td><td> Millipore </td><td> SCGPU05RE </td><td></td></tr><tr><td> 0.22 μm의, 500 ML 용량 </td><td></td><td></td><td></td></tr><tr><td> Trypan 블루 </td><td> Invitrogen </td><td> 15250061 </td><td></td></tr><tr><td> 트립신-EDTA (에틸렌 다이아 민 테트라 초산), 0.05 % (W / V) </td><td> Invitrogen </td><td> 25300-054 </td><td></td></tr><tr><td> 십대 초반-20 </td><td> 시그마 </td><td> P7949 </td><td></td></tr><tr><td> Valproic 산성 </td><td> 시그마 </td><td> P4543 </td><td></td></tr><tr><td> 접선 흐름 시스템을 Centramate </td><td> 물리다 </td><td></td><td></td></tr><tr><td> CO 2 humidified 인큐베이터, 표준 6.0 세제곱. 피트 </td><td> 다양한 브랜드가 있습니다 </td><td></td><td></td></tr><tr><td> 전기 및 전송 장치 </td><td> 다양한 브랜드가 있습니다 </td><td></td><td></td></tr><tr><td> 보육, 37 ° C </td><td> 다양한 브랜드가 있습니다 </td><td></td><td></td></tr></tbody></table>

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

세균에서 재조합 단백질 발현, 일반적으로 E. 대장균은 단백질의 밀리그램 수량 표현을위한 가장 성공적인 전략이었습니다. 그러나, prokaryotic 호스트는 종종 박테리아 특히 공동 또는 사후 translational 수정의 부족으로 인해 인간, 바이러스성 또는 진핵세포 외국 macromolecule의 독성으로 인해 단백질, 단백질 폴딩 기계의 차이, 또는 표현에 따라 않습니다. 효모 (P.의 pastoris 혹은 S. cerevisiae의) 1,2, baculovirus에 감염된 곤충 (S. frugiperda 또는 T. NI) 셀 3, 체외 번역 시스템의 셀 - 무료를 기반으로 표현 시스템은 2,4가 성공적으로 사용되었습니다 포유 동물 단백질을 생산하고 있습니다. 직관적으로, 최고의 경기는 적절한 사후 translational 개조를 포함하는 재조합 단백질의 생산을 보장하기 위해 포유류의 호스트를 사용하는 것입니다. 포유 동물 세포 라인 (인간 배아 키즈의 숫자네이 (HEK) 293은 S V40 larget T-항원 (COS), 중국 햄스터 난소 (CHO), 그리고 다른 사람을 운반 O rigin에서 C V-1 세포)가 성공적으로 인간 단백질 개수 밀리그램 수량 overexpress 위해 활용되었습니다 5-9. 그러나 포유 동물 세포를 사용의 이점은 주로 안정적인 표현 세포 라인을 개발하기 위해 높은 비용, 전문 실험실 장비의 요구 사항, 낮은 단백질 수율, 그리고 긴 시간으로 반박하고 있습니다. 적은 비용을 유지하면서, 빠른 수율 및 생산 단백질의 증가, 많은 학술 및 상업 실험실을위한 주요 요소입니다. 여기서는 시간과 점착 성의 HEK 293T 세포에서 분비 인간 단백질의 표현을위한 비용 효율적인 두 부분으로 절차를 설명합니다. 이 시스템은 구조적 biophysical 및 생화 학적 연구를위한 기능성 단백질의 밀리그램 수량에 마이크로 그램 제작이 가능합니다. 첫 번째 부분은 관심있는 유전자의 여러 구조는 생산이다병렬 및 transiently 작은 규모의 점착 성의 HEK 293T 세포로 transfected에서 D. 세포 배양 배지로 분비 재조합 단백질의 검출 및 분석은 벡터 인코딩된 단백질 정제 태그 향한 상용화된 항체를 사용하여 서양 얼룩 분석에 의해 수행됩니다. 그 후, 대규모 단백질 생산에 적합한 구조는 transiently 10 층 전지 공장에서 polyethyleneimine (PEI)를 사용 transfected 있습니다. 에어컨 매체의 리터 - 볼륨으로 분비 단백질은 안티 - HA 친화성 크로마 토그래피에 의해 정제 다음에 접선 유동 여과를 사용하여 관리 가능한 금액으로 집중되고있다. 이 플랫폼의 유틸리티는 크린 시토킨, 시토킨 수용체, 세포 표면 수용체, 본질적인 제한 요인과 바이러스 glycoproteins의 밀리그램 수량을 표현하는 기능에 의해 입증된다. 이 방법은 또한 성공적으로 trimeric ebolavirus의 당단백질의 5,10의 구조 결정에 사용되었다. 2 배양기 이외의 추가적인 장비는, 필요하지 않습니다. 이 절차는 신속하게 이러한 단백질 단지, 항원과 항체, 백신 생산을위한 바이러스 같은 입자, 또는 어려운 셀 라인 도입에 대해 adenoviruses 또는 lentiviruses 생산의 공동 표현과 같은 큰 복잡한 체제를 지원하기 위해 확장할 수 있습니다.

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

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

이후 인간 게놈 시대에 원시 conformations에서 재조합 단백질의 가용성은 구조적 기능적 및 치료 연구와 개발에 매우​​ 중요합니다. 여기서는 테스트 및 재조합 단백질의 다양한 생산하는 데 사용할 수있는 인간의 배아 신장 293T 세포에 대규모 단백질 발현 시스템을 설명합니다.

인간의 세포에서 분비 단백질의 생산을위한 편리하고 일반적인 표현 플랫폼

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. ...

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JoVE Journal

Biology

21.0K 조회수.  University of Toronto. 이후 인간 게놈 시대에 원시 conformations에서 재조합 단백질의 가용성은 구조적 기능적 및 치료 연구와 개발에 매우​​ 중요합니다. 여기서는 테스트 및 재조합 단백질의 다양한 생산하는 데 사용할 수있는 인간의 배아 신장 293T 세포에 대규모 단백질 발현 시스템을 설명합니다.

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

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 ...

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

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 ...

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.

의 효율적인 생산 및 정제 재조합 쥐과 Kindlin-3 생물 물리학 연구 곤충 세포에서

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

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

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

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

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

막 단백질의 녹색 형광 단백질 기반의 표현 상영에<em&gt; 대장균</em

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

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

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of cell proliferation, survival, migration, etc. The ECM plays important roles in development and in diverse pathologies including cardio-vascular and musculo-skeletal diseases, fibrosis, and cancer. Thus, characterizing the composition of ECMs of normal and diseased tissues could lead to the identification of novel prognostic and diagnostic biomarkers and potential novel therapeutic targets. However, the very nature of ECM proteins (large in size, cross-linked and covalently bound, heavily glycosylated) has rendered biochemical analyses of ECMs challenging. To overcome this challenge, we developed a method to enrich ECMs from fresh or frozen tissues and tumors that takes advantage of the insolubility of ECM proteins. We describe here in detail the decellularization procedure that consists of sequential incubations in buffers of different pH and salt and detergent concentrations and that results in 1) the extraction of intracellular (cytosolic, nuclear, membrane and cytoskeletal) proteins and 2) the enrichment of ECM proteins. We then describe how to deglycosylate and digest ECM-enriched protein preparations into peptides for subsequent analysis by mass spectrometry.

This protocol describes a procedure for enriching ECM proteins from tissues or tumors and deglycosylating and digesting the ECM-enriched preparations into peptides to analyze their protein composition by mass spectrometry.

질량 분석기 분석을위한 펩티드로 조직과 소화에서 세포 외 기질 단백질의 농축

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

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

The art of producing recombinant proteins with complex post-translational modifications represents a major challenge for studies of structure and function. The rapid establishment and high recovery from transiently-transfected mammalian cell lines addresses this barrier and is an effective means of expressing proteins that are naturally channeled through the ER and Golgi-mediated secretory pathway. Here is one protocol for protein expression using the human HEK293F and HEK293S cell lines transfected with a mammalian expression vector designed for high protein yields. The applicability of this system is demonstrated using three representative glycoproteins that expressed with yields between 95-120 mg of purified protein recovered per liter of culture. These proteins are the human Fc&#947;RIIIa and the rat &#945;2-6 sialyltransferase, ST6GalI, both expressed with an N-terminal GFP fusion, as well as the unmodified human immunoglobulin G1 Fc. This robust system utilizes a serum-free medium that is adaptable for expression of isotopically enriched proteins and carbohydrates for structural studies using mass spectrometry and nuclear magnetic resonance spectroscopy. Furthermore, the composition of the N-glycan can be tuned by adding a small molecule to prevent certain glycan modifications in a manner that does not reduce yield.

Laboratory-scale production of eukaryotic proteins with appropriate post-translational modification represents a significant barrier. Here is a robust protocol with rapid establishment and turnaround for protein expression using a mammalian expression system. This system supports selective amino acid, selective labeling of proteins and small molecule modulators of glycan composition.

서스펜션의 HEK293 세포의 과도 형질와 재조합 인간 단백질의 높은 수율 식

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

Transient Expression and Cellular Localization of Recombinant Proteins in Cultured Insect Cells

Heterologous protein expression systems are used for the production of recombinant proteins, the interpretation of cellular trafficking/localization, and the determination of the biochemical function of proteins at the sub-organismal level. Although baculovirus expression systems are increasingly used for protein production in numerous biotechnological, pharmaceutical, and industrial applications, nonlytic systems that do not involve viral infection have clear benefits but are often overlooked and underutilized. Here, we describe a method for generating nonlytic expression vectors and transient recombinant protein expression. This protocol allows for the efficient cellular localization of recombinant proteins and can be used to rapidly discern protein trafficking within the cell. We show the expression of four&#160;recombinant proteins in a commercially available insect cell line, including two aquaporin proteins from the insect Bemisia tabaci, as well as subcellular marker proteins specific for the cell plasma membrane and for intracellular lysosomes. All recombinant proteins were produced as chimeras with fluorescent protein markers at their carboxyl termini, which allows for the direct detection of the recombinant proteins. The double transfection of cells with plasmids harboring constructs for the genes of interest and a known subcellular marker allows for live cell imaging and improved validation of cellular protein localization.

Nonlytic insect cell expression systems are underutilized for production, cellular trafficking/localization, and recombinant protein functional analysis. Here, we describe methods to generate expression vectors and subsequent transient protein expression in commercially available lepidopteran cell lines. The co-localization of Bemisia tabaci aquaporins with subcellular fluorescent marker proteins is also presented.

배양 된 곤충 세포의 과도 표현 및 재조합 단백질의 세포 현지화

Heterologous protein expression systems are used for the production of recombinant proteins, the interpretation of cellular trafficking/localization, and ...