Este trabalho foi financiado por um Tratamento HIV Ontario Rede de Pesquisa Operacional Grant (ROG-G645) e Canadian Institutes of Health Research New Investigator Award (MSH-113554) para JEL, e da Universidade de Bolsas de Toronto para HA, FCA, e JDC. Os autores gostariam de agradecer Marnie Fusco, Dafna Abelson e Dr. Erica Ollmann Saphire no The Scripps Research Institute (La Jolla, CA) para as células que fornecem, vetor de expressão Ebolavirus GP e conselhos gerais.

<ol>
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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eukaryotic+expression:+developments+for+structural+proteomics.">Eukaryotic expression: developments for structural proteomics.</a> Acta. Crystallogr. D Biol. Crystallogr. 62, 1114-1124 (2006).</li><li>Aricescu, A. R., Lu, W., Jones, E. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+time-+and+cost-efficient+system+for+high-level+protein+production+in+mammalian+cells.">A time- and cost-efficient system for high-level protein production in mammalian cells.</a> Acta. Crystallogr. D Biol. Crystallogr. D62, 1243-1250 (2006).</li><li>Chang, V. 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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+the+quaternary+complex+of+interleukin-2+with+its+alpha,+beta,+and+gammac+receptors.">Structure of the quaternary complex of interleukin-2 with its alpha, beta, and gammac receptors.</a> Science. 310, 1159-1163 (2005).</li></ol>

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As fábricas 10-camada de células são um vaso eficaz para a produção de quantidades de miligramas de proteína. Uma grande vantagem de utilizar a fábrica celular sobre outros vasos tradicionais, tais como garrafas cilíndricas, frascos de agitação ou frascos giratórios, é que eles não requerem a aquisição de qualquer equipamento de laboratório adicional. Um padrão incubadora de CO 2 (aproximadamente 6,0 Cu. Ft) irá facilmente acomodar quatro fábricas 10 da camada de células (Fig. 2).<...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> Fosfatase alcalina (BCIP / NBT) de solução de substrato líquido </td><td> Sigma </td><td> B6404 </td><td></td></tr><tr><td> Antibiótico / antimicótico, 100X </td><td> Invitrogen </td><td> 15240062 </td><td></td></tr><tr><td> Anti-HA matriz de afinidade, o clone 3F10 </td><td> Roche </td><td> 1815016 </td><td></td></tr><tr><td> Anti-HA de murino mAb, clone 16B12 </td><td> Covance </td><td> MMS-101P </td><td></td></tr><tr><td> Balão de cultura de células, cultura de tecidos T75 centímetros 2 tratada </td><td> Corning </td><td> 430641 </td><td></td></tr><tr><td> Balão de cultura de células, T225 centímetros de cultura de tecidos 2 tratada </td><td> Corning </td><td> 431082 </td><td></td></tr><tr><td>As placas de cultura de células bem ,6-cultura de tecidos tratados </td><td> Corning </td><td> 3516 </td><td></td></tr><tr><td> Fábrica de células, CellSTACK 10 camadas </td><td> Corning </td><td> 3312 </td><td></td></tr><tr><td> Centramate Omega Cassete 5K </td><td> Mortalha </td><td> OS005C12 </td><td></td></tr><tr><td> Centramate Omega 30K Cassete </td><td> Mortalha </td><td> OS030C12 </td><td></td></tr><tr><td> Cromatografia em coluna de vidro, cm 1.0x10 </td><td> Kontes </td><td> 4204001010 </td><td></td></tr><tr><td> Ciprofloxacina </td><td> Sigma </td><td> 17850 </td><td></td></tr><tr><td> CO 2 </td><td></td><td></td><td></td></tr><tr><td> Meios Dulbecco modificado por Eagle (DMEM) </td><td> Sigma </td><td> D5796 </td><td></td></tr><tr><td> Soro fetal de bovino (FBS), inactivado pelo calor </td><td> Invitrogen </td><td> 12484-028 </td><td></td></tr><tr><td> FuGENE reagente transfecção HD </td><td> Promega </td><td> 4709691001 </td><td></td></tr><tr><td> GeneJuice reagente transfecção </td><td> EMD / Merck </td><td> 70967-6 </td><td></td></tr><tr><td> Glycine </td><td> Sigma </td><td> G8898 </td><td></td></tr><tr><td> Cabra anti-rato IgG F (ab &#39;) 2 alcalina </td><td> Thermo Scientific </td><td> 31324 </td><td></td></tr><tr><td> fosfatase anticorpo conjugado </td><td></td><td></td><td></td></tr><tr><td> Hemaglutinina (HA) péptido, 100 mg </td><td> GenScript </td><td> síntese feita sob encomenda </td><td></td></tr><tr><td> (Seqüência: YPYDVPDYA; grau de pureza 95%) </td><td></td><td></td><td></td></tr><tr><td> As células HEK 293T </td><td> ATCC </td><td> CRL-11268 </td><td></td></tr><tr><td> Household lixívia (4% w / v de hipoclorito de sódio) </td><td> várias marcas estão disponíveis </td><td></td><td></td></tr><tr><td> Immobilon-P PVDF membrana </td><td> Millipore </td><td> IPVH07850 </td><td></td></tr><tr><td> Miniprep kit de purificação de plasmídeo, PureLink rápida </td><td> Invitrogen </td><td> K2100-11 </td><td></td></tr><tr><td> MaxiPrep kit de purificação de plasmídeo, PureLink HiPure </td><td> Invitrogen </td><td> K2100-07 </td><td></td></tr><tr><td> NaN 3 </td><td> Sigma </td><td> S8032 </td><td></td></tr><tr><td> vetor de expressão pDISPLAY </td><td> Invitrogen </td><td> V660-20 </td><td></td></tr><tr><td> Penicilina / estreptomicina (pen / strep), 100X </td><td> Invitrogen </td><td> 15140-122 </td><td></td></tr><tr><td> Salino tamponado com fosfato (PBS) estéril, 1X </td><td> Sigma </td><td> D8537 </td><td></td></tr><tr><td> Polietilenoimina (PEI), lineares 25 kDa </td><td> Polyscience </td><td> 23966 </td><td></td></tr><tr><td> Leite desnatado em pó seco </td><td> Cravo </td><td></td><td></td></tr><tr><td> Stericup-GP unidade de filtração PES vácuo, </td><td> Millipore </td><td> SCGPU05RE </td><td></td></tr><tr><td> 0,22, 500 ml de capacidade </td><td></td><td></td><td></td></tr><tr><td> Trypan azul </td><td> Invitrogen </td><td> 15250061 </td><td></td></tr><tr><td> Tripsina-EDTA, 0,05% (w / v) </td><td> Invitrogen </td><td> 25300-054 </td><td></td></tr><tr><td> Tween-20 </td><td> Sigma </td><td> P7949 </td><td></td></tr><tr><td> O ácido valpróico </td><td> Sigma </td><td> P4543 </td><td></td></tr><tr><td> Centramate sistema de fluxo tangencial </td><td> Mortalha </td><td></td><td></td></tr><tr><td> Incubadora de CO 2 umidificado, padrão 6.0 cu. pé </td><td> várias marcas estão disponíveis </td><td></td><td></td></tr><tr><td> Eletroforese e transferência </td><td> várias marcas estão disponíveis </td><td></td><td></td></tr><tr><td> Incubadora, a 37 ° C </td><td> várias marcas estão disponíveis </td><td></td><td></td></tr></tbody></table>

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

A expressão da proteína recombinante em bactérias, tipicamente E. coli, tem sido a estratégia mais bem sucedido para a expressão quantidade miligrama de proteínas. No entanto, hospedeiros procarióticos não são frequentemente como apropriado para a expressão de humanos, as proteínas virais ou eucarióticos, devido à toxicidade da macromolécula externa, as diferenças de a máquina de dobragem de proteína, ou devido à falta de modificações específicas co ou pós-translacional em bactérias. Os sistemas de expressão baseados em levedura (P. pastoris ou S. cerevisiae) 1,2, baculovírus-infectada de insectos (S. frugiperda ou T. ni), as células 3, e livre de células in vitro tradução sistemas 2,4 foram utilizados com sucesso para produzir proteínas de mamíferos. Intuitivamente, a melhor correspondência é usar um hospedeiro mamífero para assegurar a produção de proteínas recombinantes que contêm as adequadas modificações pós-translacionais. Um certo número de linhas de células de mamíferos (Kid embrionário humanoNey (HEK) 293, C V-1 em células que transportam o O RIGEM S V40 larget antigénio T-(COS), de Ovário de Hamster Chinês (CHO), e outros) têm sido utilizados com sucesso para superexpressar quantidades de miligramas de um número de proteínas humanas 5-9. No entanto, as vantagens da utilização de células de mamíferos são frequentemente contrariada pela custos mais elevados, a exigência de equipamento de laboratório especializado, rendimentos mais baixos de proteína, e os tempos longos para desenvolver linhagens de células estáveis ​​de expressão. Aumento de produtividade e produção de proteínas mais rápido, mantendo os custos baixos, são fatores importantes para muitos laboratórios acadêmicos e comerciais. Aqui, nós descrevemos um tempo e custo-eficácia, o procedimento de duas partes para a expressão de proteínas secretadas humanos a partir de células aderentes HEK 293T. Este sistema é capaz de produzir quantidades de micrograma miligrama de proteína funcional para estudos estruturais, biofísicas e bioquímicas. A primeira parte, as construções de múltiplas do gene de interesse são produzird em paralelo e transientemente transfectados para células aderentes HEK 293T em pequena escala. A detecção e análise de proteína recombinante segregada no meio de cultura de células é realizada por análise de Western blot utilizando anticorpos comercialmente disponíveis dirigidos contra uma etiqueta de purificação do vector proteína codificada. Subsequentemente, as construções adequadas para produção em larga escala de proteínas são transitoriamente transfectadas utilizando polietilenoimina (PEI) em fábricas 10 da camada de células. As proteínas segregadas no litros de volumes de meio condicionado são concentrados em quantidades administráveis ​​usando filtração de fluxo tangencial, seguido de purificação por anti-HA cromatografia de afinidade. A utilidade desta plataforma é comprovada pela sua capacidade de expressar quantidades de miligramas de citocinas, receptores de citocinas, receptores da superfície celular, os factores de restrição intrínsecas, e glicoproteínas virais. Este método foi também utilizada com sucesso na determinação estrutural das 5,10 da glicoproteína triméricos Ebolavirus. <p class = &quot;jove_content&quot;&gt; Em conclusão, esta plataforma oferece facilidade de uso a velocidade e escalabilidade, maximizando a qualidade da proteína e funcionalidade. Além disso, nenhum equipamento adicional, que não seja um padrão humidificada incubadora de CO 2, é necessário. Este procedimento pode ser rapidamente expandido para sistemas de maior complexidade, tais como co-expressão de complexos de proteína, antigénios e anticorpos, produção de partículas semelhantes a vírus para as vacinas, ou a produção de adenovírus ou lentivírus para transdução de linhas de células difíceis.

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

Na era pós-genômica humana, a disponibilidade de proteínas recombinantes em conformações nativas é crucial para a investigação estrutural, funcional e terapêutico e desenvolvimento. Aqui, nós descrevemos um-teste e em grande escala sistema de expressão de proteínas em células humanas embrionárias de rim 293T que podem ser utilizados para produzir uma variedade de proteínas recombinantes.

A Plataforma de expressão adequada e geral para a produção de proteínas secretadas a partir de células humanas

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

a-convenient-general-expression-platform-for-production-secreted

Research

JoVE Journal

Biology

21.0K visualizações.  University of Toronto. Na era pós-genômica humana, a disponibilidade de proteínas recombinantes em conformações nativas é crucial para a investigação estrutural, funcional e terapêutico e desenvolvimento. Aqui, nós descrevemos um-teste e em grande escala sistema de expressão de proteínas em células humanas embrionárias de rim 293T que podem ser utilizados para produzir uma variedade de proteínas recombinantes.

Vídeo: 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.

Elaboração de culturas de células primárias hematopoéticas de medula óssea de camundongos para Eletroporação

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

Biologia

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.

Coloração de proteínas em géis com Coomassie G-250 sem ácido acético e Solventes Orgânicos

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.

Usando o Sistema de Eletroporação Gene Pulser MXcell para transfectar pilhas com Alta Eficiência

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.

Usando um contador de células automatizadas para simplificar Estudos da Expressão Gênica: Knockdown siRNA de Expressão IL-4 Gene Dependente em células NAMALWA

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.

A Plataforma de Produção de Proteína Complexo MultiBac no EMBL

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.

Produção eficiente e Purificação de recombinante murino Kindlin-3 a partir de células de insetos de Estudos biofísicos

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.

Verde Fluorescente à base de proteínas Expressão Rastreio de Proteínas de Membrana em<em&gt; Escherichia coli</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.

Enriquecimento de Proteínas da Matriz Extracelular a partir de tecidos e digestão em péptidos para Análise de Espectrometria de Massa

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.

High Yield Expressão de proteínas recombinantes humanas com o Transient transfecção de células HEK293 em suspensão

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.

Expressão transitória e Celular Localização de proteínas recombinantes em células de cultura de insectos

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