I. Descongelar las células Tome el frasco de helado de TN5 las células, y descongelar en un baño de agua a 37 ° C. Añadir 5 ml de medio de insectos Express (Invitrogen, número de catálogo-10486) en el frasco de 25 cm TC 2. Tenga en cuenta que Express cinco viene sin glutamina, necesita agregar 10 ml de estreptococos pluma 100X glutamina o glutamina solo a 1 lt medios de comunicación. Añadir TN5 células en ella y se incuba durante 30 minutos en una incubadora de 27 ° C. Luego, se aspira el medio con DMSO suavemente sin molestar a las células unidas al fondo del frasco. Añadir 5 ml de medio fresco. II. Crecimiento y expansión de las células Monitorizar las células todos los días. Cuando frasco es de casi el 70% confluentes, llevar a las células en suspensión por golpear el frasco en ambos lados y la transferencia de las células de 175cm 2 frasco mediante la adición de 21 ml de insectos a medio expresar y 5 ml de cultivo celular. Cada frasco T175 debe tener alrededor de 25-30ml medio como un volumen final. Dividir frasco confluente (aproximadamente 1 millón / ml conc.) 1 / 4 o 1 / 5, según sea necesario. No deje crecer demasiado las células. Cuente el número de pasajes que se han separado de las células. No crecen en el paso número 30, ya que puede causar el envejecimiento de las células resultantes lisis celular. Al alcanzar el volumen deseado en la concentración de 1 millón / ml, infectar las células. Yo por lo general crecen de 2 a 2,5 litros, que por lo general asciende a setenta y dos frascos de 175 cm y aprox. 25 a 30 ml frasco /, o cinco matraces Erlenmeyer de 1 litro y aprox. 400 ml a 500 ml cada frasco Nota: puede crecer en las células T-175 frasco de sello de tapón o matraz Erlenmeyer Corning. Crecimiento de las células en matraces Erlenmeyer es más rápido y más fácil. III. Titulación del virus por el método de dilución de punto final <ol><li> Placa de 3000-5000 células por pozo en el fondo plano placa de 96 pocillos en 200ul de medio expreso SFM cinco. Vamos a colocar las células a 27 ° C durante al menos 30 minutos. </li><li> Hacer series de dilución de las acciones de baculovirus. Uno debe hacer dilución 1 / 10 en una línea de 96 y placa inferior U, con 250ul por pocillo. Diluciones se hacen en medio de la ordenación forestal sostenible expreso Cinco. </li><li> Con una pipeta multicanal, la transferencia de una cantidad fija (por lo general 20 ul) de diferentes diluciones de las células. </li><li> La cultura a 27 ° C durante 7 días. Para evitar la evaporación de las filas de borde, envuelva los bordes de la placa con parafilm. </li><li> Después de 7 días, verifique la placa y determinar la dilución de virus que da una infección en un 50% - la mitad de los pozos en esta concentración de virus están infectados). A continuación, utilice una fórmula para calcular el título, que se expresa en ufp / ml (PFU-unidades formadoras de placa). La fórmula se encuentra en los manuales de la mayoría de los baculovirus. </li></ol> IV. Infectando las células Es muy importante saber el título exacto del virus. Añadir la cantidad necesaria de virus. Para la preparación del stock viral de las células deben estar infectadas a una multiplicidad de infección (MOI), de no más de 1 (1pfu a 1High Cinco celular) MOI superior lleva a la producción de mutaciones virales. Para la producción de proteínas, la cantidad normal es de 10.5 MO1. Ejemplo: El título de baculovirus mCD1d = 1x108 ufp / ml Añadir MOI = 1 (para amplificar el virus) = 20X10 ^ 6 células / frasco / 1x10 ^ 8 ufp / ml = 200 ul / frasco MOI = 5 a 10 (para la producción de proteínas) = ​​1 ml a 2 ml por 20x10 ^ 6 células / frasco El día 4 después de la infección, mirar las células infectadas. Deben ser individual, salchichas flotantes, se inflaman y forman. V. Recuperación de los sobrenadantes Tomar el medio de los frascos de infectados, y la transferencia de 250 ml botellas de color azul tapa Falcon Spinning. Pueden ser esterilizados en autoclave y reutilizado. Llenar las botellas de manera uniforme y centrifugar las células en Sorvall GSA rotor a 200 rpm, 20 minutos, 4 ° C. Recoger el sobrenadante y proceder para una mayor purificación. VI. Purificación de proteínas recombinantes mCD1d <ol><li> Diálisis y concentración en tampón fosfato de sodio 150 mM (pH 7,4) </li><li> De Ni-NTA agarosa cromatografía, eluir la proteína mCD1d + b2m con 150 mM de fosfato sódico 200 mM Immidazole </li><li> Ejecutar columna de la desalación con 20 mM Tris-HCl pH 8 buffer para deshacerse de Immidazole </li><li> Ejecutar cromatografía de intercambio aniónico con columna MonoQ para eliminar los contaminantes restantes. </li><li> Concentrado a 1mg/ml utilizando YM 30 concentrador (Millipore) </li><li> Biotynlation BirA enzimática de mCD1d acuerdo con el protocolo del fabricante (avidez) </li><li> La biotina libre se elimina por la columna S200 (Chromatograpy de exclusión de tamaño) con tampón fosfato salino (PBS), pH 7,4. Un promedio de 2 a 3 mg de proteína biotynlated se podría obtener de dos litros de sobrenadante </li><li> Producción de un tetrámero-GalCer CD1d. </li></ol> Para cargar molécula CD1d con un GalCer, soluble biotynlated CD1d-b2m se incubaron durante la noche a temperatura ambiente, con tres veces superior a molar de un GalCer, disuelto en Tween 0,5% y el 20 en PBS, O.9% de cloruro de sodio. Tetrámeros se generan por la mezcla de uno-GalCer monómeros cargados CD1d con 4 veces superior a molar del PE - estreptavidina conjugada y la incubación durante 4 horas a temperatura ambiente. Almacenar a 48 ° C.

<ol>
	<li>Benlagha, K., Weiss, A., Beavis, A., Teyton, L., Bendelac, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+identification+of+glycolipipd+antigen-specific+T+cells+using+fluorecent+CD1d+tetramers.">In vivo identification of glycolipipd antigen-specific T cells using fluorecent CD1d tetramers.</a> J. Exp. Med. 191, 1895-1903 (2000).</li><li>Kinjo, Y., Wu, D., Kim, G., Xing, G. W., Poles, M. A., Ho, D. D., Tsuji, M., Kawahara, K., Wong, C. H., Kronenberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recognition+of+bacterial+glycosphingolipids+by+natural+killer+T+cells.">Recognition of bacterial glycosphingolipids by natural killer T cells.</a> Nature. 434, 520-525 (2005).</li><li>Naidenko, O. V., Maher, J. K., Ernst, W. A., Sakai, T., Modlin, R. L., Kronenberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Binding+and+antigen+presentation+of+ceramide-containing+glycolipids+by+soluble+mouse+and+human+CD1d+molecules.">Binding and antigen presentation of ceramide-containing glycolipids by soluble mouse and human CD1d molecules.</a> J. Exp. Med. 190, 1069-1080 (1999).</li><li>Porcelli, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+CD1+family:+A+third+lineage+of+antigen-+presenting+molecules.">The CD1 family: A third lineage of antigen- presenting molecules.</a> Adv. Immunol. 59, 1-98 (1995).</li><li>Sidobre, S., Kronemberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD1+tetramers:+A+powerful+tool+for+the+analysis+of+glycolipid-reactive+T+cells.">CD1 tetramers: A powerful tool for the analysis of glycolipid-reactive T cells.</a> J. Immunol. 169, 1340-1348 (2002).</li></ol>

En este procedimiento, se muestra cómo iniciar, crecer, infectar células de insecto y cómo el título de la baculovirus uso de estas células. Los aspectos más importantes de este procedimiento son el mantenimiento de las células y saber el título exacto de baculovirus. Una vez que se han generado CD1 tetrámeros, que puede ser utilizado como una herramienta poderosa para el análisis de glicolípido las células T reactivas. Sistemas de baculovirus también se utilizan ampliamente para producir proteínas recombinantes, incluyendo tetrámeros de MHC de clase II por lo que este procedimiento tiene una aplicación mucho más amplia.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Express Five SFM</td>
<td> Serum Free Medium</td>
<td>Invitrogen</td>
<td>10486025</td>
<td>&nbsp;</td>
<tr>
<td>High Five TM</td>
<td> Insect Cells</td>
<td>Invitrogen</td>
<td>B855-02</td>
<td>&nbsp;</td>
<tr>
<td>Glutamine Pen strep</td>
<td>Antibiotics</td>
<td>Invitrogen</td>
<td>10378-016</td>
<td>&nbsp;</td>
<tr>
<td>25cm2 TC Flask</td>
<td> Plastic</td>
<td>Fisher</td>
<td>10-126-28</td>
<td>&nbsp;</td>
<tr>
<td>175 cm2 TC Flask</td>
<td> Plug seal flask</td>
<td>Fisher</td>
<td>10-126-8</td>
<td>&nbsp;</td>
<tr>
<td>Erlenmeyer Flask</td>
<td> Cell Culture Flask</td>
<td>Fisher</td>
<td>07 200 672</td>
<td>&nbsp;</td>
<tr>
<td>YM 30 Concenterator</td>
<td>30,000 MWCO</td>
<td>Millipore</td>
<td>UFC 903096</td>
<td>&nbsp;</td>
<tr>
<td>FPLC equipment</td>
<td>&nbsp;</td>
<td>GE Healthcare</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>BrA Enzyme and Buffer</td>
<td> Biotinylation Kit</td>
<td>Avidity</td>
<td>BIRA500</td>
<td>&nbsp;</td>
<tr>
<td>Ni-NTA Agarose Column</td>
<td>His Tag Protein Purification Column</td>
<td>GE Healthcare</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Desalting column</td>
<td> To get Rod of Salt from Protein</td>
<td>GE Healthcare</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>MonoQ Column</td>
<td>Anion Exchange Chromatgraphy</td>
<td>GE Healthcare</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>S200 Column</td>
<td> Size Exclusion Chromatography</td>
<td>GE Healthcare</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>alpha-Galcer</td>
<td> Glycolipid</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>PE- Streptavidin</td>
<td> For conjugating Protein</td>
<td>Molecular Probe</td>
<td> S-866</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

a method for production of recombinant mcd1d protein in insect cells.

CD1 proteínas constituyen una tercera clase de moléculas presentadoras de antígeno. Son glicoproteínas de superficie celular, expresado en aproximadamente 50 kDa glicosilada cadenas pesadas que son covalentemente asociados con beta2-microglobulina. Se unen los lípidos en lugar de péptidos. A pesar de su estructura confirma la similitud de las proteínas CD1 a MHC de clase I y clase II que presentan las moléculas de antígeno, la ranura mCD1d es relativamente estrecha, profunda y altamente hidrofóbica y tiene dos bolsillos vinculante en lugar de los bolsillos de varios superficiales descritas para las clásicas MHC- codifican moléculas presentadoras de antígeno. Con base en sus secuencias de aminoácidos, como un surco hydrobphobic proporciona un ambiente ideal para la unión de antígenos lipídicos. El T asesinas naturales (NKT), las células utilizan su TCR de reconocer glicolípidos obligado o presentados por CD1d. Las células T reactivas a los lípidos presentados por CD1 han estado involucrados en la protección contra las enfermedades infecciosas y autoinmunes y el rechazo del tumor. Por lo tanto, la capacidad de identificar, purificar y hacer un seguimiento de la respuesta de CD1-reactiva de células NKT es de gran importancia. La generación de tetrámeros de alfa galactosil ceramida (a-GalCer) con CD1d tiene una visión significativa sobre la biología de las células NKT. Tetrámeros construido a partir de otras moléculas CD1 también se han generado y los nuevos reactivos han expandido enormemente el conocimiento de las funciones de las células T reactivas de lípidos, con uso potencial en el control de la respuesta a las vacunas basadas en lípidos y en el diagnóstico de enfermedades autoinmunes y otras los tratamientos.

Watch this Scientific Journal Video about A Method For Production of Recombinant mCD1d Protein in Insect Cells. at JoVE.com

A Method For Production of Recombinant mCD1d Protein in Insect Cells.

Un método para preparar las células de insectos y las infectan con el baculovirus con el propósito de la producción de proteínas recombinantes proteinand mCD1d generar tetrámeros mCD1d.

Un método para la producción de proteínas recombinantes en células de insecto mCD1d.

CD1 proteins constitute a third class of antigen-presenting molecules. They are cell surface glycoproteins, expressed as approximately 50-kDa glycosylated ...

a-method-for-production-of-recombinant-mcd1d-protein-in-insect-cells

Nanyang Technological University

Research

JoVE Journal

Biology

10.7K vistas.  La Jolla Institute for Allergy and Immunology. Un método para preparar las células de insectos y las infectan con el baculovirus con el propósito de la producción de proteínas recombinantes proteinand mCD1d generar tetrámeros mCD1d.

Video: A Method For Production of Recombinant mCD1d Protein in Insect Cells.

Pyrosequencing: A Simple Method for Accurate Genotyping

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a tremendous amount of data available comes an explosion of genotyping methods. Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. It also has the ability to identify tri-allelic, indels, short-repeat polymorphisms, along with determining allele percentages for methylation or pooled sample assessment. In addition, there is a standardized control sequence that provides internal quality control. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.


Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.

Pyrosequencing: Un método simple para determinar el genotipo exacto

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a ...

Investigación

Biología

A Protocol for Phage Display and Affinity Selection Using Recombinant Protein Baits

Using recombinant phage as a scaffold to present various protein portions encoded by a directionally cloned cDNA library to immobilized bait molecules is an efficient means to discover interactions. The technique has largely been used to discover protein-protein interactions but the bait molecule to be challenged need not be restricted to proteins. The protocol presented here has been optimized to allow a modest number of baits to be screened in replicates to maximize the identification of independent clones presenting the same protein. This permits greater confidence that interacting proteins identified are legitimate interactors of the bait molecule. Monitoring the phage titer after each affinity selection round provides information on how the affinity selection is progressing as well as on the efficacy of negative controls. One means of titering the phage, and how and what to prepare in advance to allow this process to progress as efficiently as possible, is presented. Attributes of amplicons retrieved following isolation of independent plaque are highlighted that can be used to ascertain how well the affinity selection has progressed. Trouble shooting techniques to minimize false positives or to bypass persistently recovered phage are explained. Means of reducing viral contamination flare up are discussed.

Phage display is a powerful technique to capture proteins or protein moieties that interact with an immobilized molecule of interest. Once a decision of the type of phage cDNA library to create and screen has been made, the protocol described here permits efficient affinity selection leading to identification of interactors.

Un Protocolo de fagos y de selección de afinidad utilizando cebos proteína recombinante

Using  recombinant phage as a scaffold to present various protein portions encoded by  a directionally cloned cDNA library to immobilized bait molecules ...

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short period of time. However, this technology is only just beginning to be applied to large-scale production as many technological obstacles to scale up are now being overcome. Here, we demonstrate a simple and reproducible method for industrial-scale transient protein production based on vacuum infiltration of Nicotiana plants with Agrobacteria carrying launch vectors. Optimization of Agrobacterium cultivation in AB medium allows direct dilution of the bacterial culture in Milli-Q water, simplifying the infiltration process. Among three tested species of Nicotiana, N. excelsiana (N. benthamiana &#215; N. excelsior) was selected as the most promising host due to the ease of infiltration, high level of reporter protein production, and about two-fold higher biomass production under controlled environmental conditions. Induction of Agrobacterium harboring pBID4-GFP (Tobacco mosaic virus-based) using chemicals such as acetosyringone and monosaccharide had no effect on the protein production level. Infiltrating plant under 50 to 100 mbar for 30 or 60 sec resulted in about 95% infiltration of plant leaf tissues. Infiltration with Agrobacterium laboratory strain GV3101 showed the highest protein production compared to Agrobacteria laboratory strains LBA4404 and C58C1 and wild-type Agrobacteria strains at6, at10, at77 and A4. Co-expression of a viral RNA silencing suppressor, p23 or p19, in N. benthamiana resulted in earlier accumulation and increased production (15-25%) of target protein (influenza virus hemagglutinin).

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

Transient protein production in Nicotiana plants based on vacuum infiltration with Agrobacteria carrying launch vectors (Tobacco mosaic virus-based) is a rapid and economic approach to produce vaccine antigens and therapeutic proteins. We simplified the procedure and improved target accumulation by optimizing conditions of bacteria cultivation, selecting host species, and co-introducing RNA silencing suppressors.

La optimización y utilización de<em&gt; Agrobacterium</em&gt; Mediada por la producción de proteína transitoria en<em&gt; Nicotiana</em

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short ...

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.

Producción eficiente y purificación de recombinante murino kindlin-3 a partir de células de insecto para estudios biofísicos

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

Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live cells. BRET is the non-radiative transfer of energy from a 'donor' luciferase enzyme to an 'acceptor' fluorescent protein. In the most common configuration of this assay, the donor is Renilla reniformis luciferase and the acceptor is Yellow Fluorescent Protein (YFP). Because the efficiency of energy transfer is strongly distance-dependent, observation of the BRET phenomenon requires that the donor and acceptor be in close proximity. To test for an interaction between two proteins of interest in cultured mammalian cells, one protein is expressed as a fusion with luciferase and the second as a fusion with YFP. An interaction between the two proteins of interest may bring the donor and acceptor sufficiently close for energy transfer to occur. Compared to other techniques for investigating protein-protein interactions, the BRET assay is sensitive, requires little hands-on time and few reagents, and is able to detect interactions which are weak, transient, or dependent on the biochemical environment found within a live cell. It is therefore an ideal approach for confirming putative interactions suggested by yeast two-hybrid or mass spectrometry proteomics studies, and in addition it is well-suited for mapping interacting regions, assessing the effect of post-translational modifications on protein-protein interactions, and evaluating the impact of mutations identified in patient DNA.

Interactions between proteins are fundamental to all cellular processes. Using Bioluminescence Resonance Energy Transfer, the interaction between a pair of proteins can be monitored in live cells and in real time. Furthermore, the effects of potentially pathogenic mutations can be assessed.

La investigación de las interacciones proteína-proteína en las células vivas utilizando la transferencia de energía de resonancia de bioluminiscencia

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live ...

Recombinant Protein Expression for Structural Biology in HEK 293F Suspension Cells: A Novel and Accessible Approach

The expression and purification of large amounts of recombinant protein complexes is an essential requirement for structural biology studies. For over two decades, prokaryotic expression systems such as E. coli have dominated the scientific literature over costly and less efficient eukaryotic cell lines. Despite the clear advantage in terms of yields and costs of expressing recombinant proteins in bacteria, the absence of specific co-factors, chaperones and post-translational modifications may cause loss of function, mis-folding and can disrupt protein-protein interactions of certain eukaryotic multi-subunit complexes, surface receptors and secreted proteins. The use of mammalian cell expression systems can address these drawbacks since they provide a eukaryotic expression environment. However, low protein yields and high costs of such methods have until recently limited their use for structural biology. Here we describe a simple and accessible method for expressing and purifying milligram quantities of protein by performing transient transfections of suspension grown HEK (Human Embryonic Kidney) 293F cells.

The expression of recombinant proteins by mammalian systems is becoming an attractive method for producing protein complexes for structural biology. Here we present a simple yet highly efficient expression system using suspension grown mammalian cells to purify protein complexes for structural studies.

Expresión de proteínas recombinantes para la Biología Estructural en HEK 293F Suspensión Células: Un enfoque novedoso y accesible

The expression and purification of large amounts of recombinant protein complexes is an essential requirement for structural biology studies. For over two ...

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.

Fluorescente verde a base de proteína Expresión Proyección de proteínas de membrana en<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 ...

A Comparative Analysis of Recombinant Protein Expression in Different Biofactories: Bacteria, Insect Cells and Plant Systems

Plant-based systems are considered a valuable platform for the production of recombinant proteins as a result of their well-documented potential for the flexible, low-cost production of high-quality, bioactive products.
In this study, we compared the expression of a target human recombinant protein in traditional fermenter-based cell cultures (bacterial and insect) with plant-based expression systems, both transient and stable.
For each platform, we described the set-up, optimization and length of the production process, the final product quality and the yields and we evaluated provisional production costs, specific for the selected target recombinant protein.
Overall, our results indicate that bacteria are unsuitable for the production of the target protein due to its accumulation within insoluble inclusion bodies. On the other hand, plant-based systems are versatile platforms that allow the production of the selected protein at lower-costs than Baculovirus/insect cell system. In particular, stable transgenic lines displayed the highest-yield of the final product and transient expressing plants the fastest process development. However, not all recombinant proteins may benefit from plant-based systems but the best production platform should be determined empirically with a case-by-case approach, as described here.

In this study the expression of a target human recombinant protein in different production platforms was compared. We focused on traditional fermenter-based cultures and on plants, describing the set-up of each system and highlighting, on the basis of the reported results, the inherent limits and advantages for each platform.

Un Análisis Comparativo de Expresión recombinante de proteínas en diferentes Biofactorías: Las bacterias, células de insecto y Plant Systems

Plant-based systems are considered a valuable platform for the production of recombinant proteins as a result of their well-documented potential for the ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

Nefrotoxina Microinyección en pez cebra para modelar la lesión renal aguda

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

Una interfaz gráfica de usuario para el seguimiento asistido por software de la concentración de proteínas en protones dinámicos celulares

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...