Este proyecto fue parcialmente financiado por un IOS NSF (0849230); Hatch, McIntire-Stennis (AD421 CRIS), Fondo Semilla USDA (2011 a 04375), y Sir Frederick McMaster de Becas de Investigación de ABD.

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Los autores no tienen nada que revelar.

Mediante la ejecución del experimento en tres pocillos replicados, fago adquirido de forma independiente de la misma proteína de unión al cebo puede distinguirse incluso si son del mismo clon (es decir, no hay diferencia en la secuencia de nucleótidos de la región de CDS que ha sido adquirida, pero estos han sido recuperados de pozos independientes). De lo contrario, la única manera de distinguir entre el fago que codifica la misma proteína de unión a la carnada es si son independientemente inversa regi...

¿Por qué utilizar la presentación en fagos y selección por afinidad en vez de las otras técnicas disponibles para miles de descubrir e investigar las interacciones de proteínas con otras moléculas? La presentación en fagos puede reclamar algunas ventajas únicas sobre otros métodos de detección de interacciones proteína-ligando de 1-3, incluyendo lo siguiente: Muy amplio repertorio de moléculas de cebo La razón más importante es en la diversidad de moléculas capaces de actuar como cebo en la selección por afinidad 4. La presentación en fagos es un medio muy poderoso para aislar fragmentos de proteínas que interactúan con otras proteínas, nucleótidos, carbohidratos, etc 5 Esencialmente, si un polímero / molécula puede estar unido a un soporte recuperable, que puede ser examinado para afinidad con las proteínas mostradas de fagos. Además, la molécula de cebo se puede acceder para determinar si se retiene la actividad biológica cuando se inmoviliza 6, si las condiciones utilizadas para reducir / eliminar su actividad son eficaces o 6, de introducir modificaciones después de la traducción a la carnada antes de la selección por afinidad. Resistencia a los fagos a factores externos Una segunda razón para el uso de presentación en fagos, es que es posible que algunos cebos pueden requerir estrés ambiental (calor, altas concentraciones osmolíticas, cofactores específicos, etc.) Para capturar sus proteínas que interactúan, o las proteínas que se muestran fagos puede necesitar ser alterado de alguna manera antes de la selección por afinidad. El factor principal que se está estudiando es la interacción entre la proteína cebo y, no la condición de que permite la interacción que se produzca o si es letal para el organismo de prueba. El fago T7 está particularmente bien adaptado a este tipo de estudios, ya que puede soportar condiciones experimentales duras tanto intactas y viables (por ejemplo, el máximo térmico publicado para la viabilidad de T7 es de ~ 60 ° C 7). Como un ejemplo de fago que altera la muestra Proproteínas, al examinar el proteoma de semillas de Arabidopsis thaliana para sustratos de proteína de la enzima reparadora, Proteína Isoaspartyl metil transferasa (PIMT), el virus usado en estos estudios habían sido &quot;abierto&quot; durante una semana, antes de cada ronda de selección por afinidad, para fomentar la introducción de isoaspartate (isoAsp) los residuos en las proteínas susceptibles 6 que no es posible en los organismos que pueden reconocer y reparar / metabolizar tales anomalías. Metabólicamente inerte Además, el fago son generalmente resistentes a venenos metabólicos y interferir moléculas pequeñas que, por lo menos, dar lugar a efectos pleiotrópicos sobre los organismos de prueba metabólicamente activas. Tras los lavados de rigurosidad, el veneno se elimina antes de que un gran volumen de bacterias se introducen para la infección de modo que el veneno se diluye a una gama inocuo para las bacterias o la replicación del fago posterior. Mientras investiga dianas proteicas de la reparación PIMTenzima, S-adenosil metionina (AdoMet) se utiliza para activar la enzima de microtitulación-placa-así inmovilizado para permitir la captura de la proteína diana mientras que confían en S-adenosil homocisteína (AdoHcy) para inactivar la enzima y proporcionar un control negativo útil en asegurar el conocimiento de que el virus no se vería influenciada negativamente por cualquiera AdoMet o AdoHcy 6. Además, los miembros de ciertas familias de la embriogénesis tardía abundante de proteínas (LEA), investigados en este laboratorio, se sabe que altera su forma en presencia de aditivos tales como sacarosa 8, que pueden alcanzar concentraciones de hasta 200 mm de semillas de soja en el punto de madurez fisiológica 9. No se prevé que el virus sea influenciado por adición de sacarosa 200 mM en cada ronda de selección por afinidad, posiblemente necesario para determinadas interacciones de proteínas de LEA-cliente, que no es el caso de organismos de ensayo viables autónoma 10. Esta práctica de laboratorio se ha centrado en DESCUBRIRg interacciones proteína-proteína en semillas maduras, deshidratados o en germinación que subyacen en el mecanismo de protección proteoma almacenado durante la deshidratación 11 o la reparación de componentes de la proteoma almacenado que son susceptibles a la formación isoAsp una vez que la semilla ha embebido 6. Por lo tanto, la producción y purificación de las proteínas recombinantes requeridos como cebo en un estado activo, y la garantía de que se mantengan así, antes y después de que se inmovilizan, aunque con frecuencia difícil, es una piedra angular de nuestro trabajo. Sin embargo, a medida que cada escenario de producción de proteínas recombinantes es diferente, las condiciones para la optimización de la producción de proteína recombinante no se abordan aquí. Se insta a los usuarios a intentar, siempre que sea posible, para determinar si la proteína inmovilizada es aún funcional (por ejemplo, si se trata de una enzima, hacer un ensayo enzimático en los pocillos de la placa de microtitulación). Esto proporciona cierta confianza en que el cebo es biológicamente activo y por lo tanto, que cualquier interacción descubiertos sonalgo más propensos a tener relevancia biológica.

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Tryptone</td>
 <td>Becton Dickinson Co.</td>
 <td>211705</td>
 <td></td>
 </tr>
 <tr>
 <td>Yeast Extract</td>
 <td>Becton Dickinson</td>
 <td>212750</td>
 <td></td>
 </tr>
 <tr>
 <td>Agar</td>
 <td>Becton Dickinson</td>
 <td>214010</td>
 <td></td>
 </tr>
 <tr>
 <td>Sodium Chloride</td>
 <td>Fisher Scientific</td>
 <td>BP358-10</td>
 <td></td>
 </tr>
 <tr>
 <td>Agarose</td>
 <td>Research Products International</td>
 <td>A20090</td>
 <td></td>
 </tr>
 <tr>
 <td>Blocking Reagent</td>
 <td>EMD Chemicals Inc.</td>
 <td>69064</td>
 <td></td>
 </tr>
 <tr>
 <td>Tween 20</td>
 <td>Fisher Scientific</td>
 <td>BP337-100</td>
 <td></td>
 </tr>
 <tr>
 <td>Sodium Hydroxide</td>
 <td>Fisher Scientific</td>
 <td>BP359-212</td>
 <td></td>
 </tr>
 <tr>
 <td>Sodium Dodecyl Sulfate</td>
 <td>Fisher Scientific</td>
 <td>BP166-500</td>
 <td></td>
 </tr>
 <tr>
 <td>Tris Base</td>
 <td>Fisher Scientific</td>
 <td>BP152-5</td>
 <td></td>
 </tr>
 <tr>
 <td>Chloroform</td>
 <td>Fisher Scientific</td>
 <td>BP1145-1</td>
 <td></td>
 </tr>
 <tr>
 <td>Escherichia coli BLT5403</td>
 <td>EMD Chemicals Inc.</td>
 <td>BLT5403</td>
 <td>Genotype: F- ompT hsdSB (rB - mB -) gal dcm pAR5403 (AmpR)</td>
 </tr>
 <tr>
 <td>ampicillin, Sodium Salt</td>
 <td>Research Products International</td>
 <td>A40040</td>
 <td></td>
 </tr>
 <tr>
 <td>ethidium bromide</td>
 <td>Fisher Scientific</td>
 <td>BP102-1</td>
 <td></td>
 </tr>
 <tr>
 <td>Bovine Serum Albumin (BSA)</td>
 <td>Sigma-Aldrich Corp.</td>
 <td>A-9647</td>
 <td></td>
 </tr>
 <tr>
 <td>penta-HIS primary antibody</td>
 <td>Qiagen Inc.</td>
 <td>34660</td>
 <td></td>
 </tr>
 <tr>
 <td>Goat anti mouse alkaline phosphatise conjugate</td>
 <td>Sigma-Aldrich Corp.</td>
 <td>A-5153</td>
 <td></td>
 </tr>
 <tr>
 <td>para-nitrophenylphosphate</td>
 <td>Sigma-Aldrich Corp.</td>
 <td>N-7653</td>
 <td></td>
 </tr>
 <tr>
 <td>T7-UP primer</td>
 <td>EMD Chemicals Inc.</td>
 <td></td>
 <td>5'-GGAGCTGTCGTATTCCAGTC-3'</td>
 </tr>
 <tr>
 <td>T7-DOWN primer</td>
 <td>EMD Chemicals Inc.</td>
 <td></td>
 <td>5'-AACCCCTCAAGACCCGTTTA-3'</td>
 </tr>
 <tr>
 <td>Qiaquick PCR Purification kit</td>
 <td>Qiagen Inc.</td>
 <td>28104</td>
 <td></td>
 </tr>
 <tr>
 <td>Qiaquick Gel Extraction kit </td>
 <td>Qiagen Inc.</td>
 <td>28706</td>
 <td></td>
 </tr>
 <tr>
 <td>Big Dye Terminator v3.1 Cycle Sequencing Kit</td>
 <td>Invitrogen Life Technologies</td>
 <td>4336917</td>
 <td></td>
 </tr>
 <tr>
 <td>Heating Block</td>
 <td>Pierce Chemical Co. (Thermo Fisher Scientific Co.)</td>
 <td>18780</td>
 <td></td>
 </tr>
 <tr>
 <td>96 Well Cell Culture Plates</td>
 <td>Corning</td>
 <td>Costar 3590</td>
 <td></td>
 </tr>
 <tr>
 <td>Isotemp Incubator Oven</td>
 <td>Fisher Scientific</td>
 <td>516D</td>
 <td></td>
 </tr>
 <tr>
 <td>Pasteur Pipets, 5 &frac34;"</td>
 <td>Fisher Scientific</td>
 <td>13-678-20A</td>
 <td></td>
 </tr>
 <tr>
 <td>ChromaView Transilluminator</td>
 <td>UVP Inc.</td>
 <td>TS-15</td>
 <td></td>
 </tr>
 <tr>
 <td>UV Shield</td>
 <td>Oberon</td>
 <td>071AF</td>
 <td></td>
 </tr>
 <tr>
 <td>Gloves, Nitrile</td>
 <td>Fisher Scientific</td>
 <td>19-170-010C</td>
 <td></td>
 </tr>
 <tr>
 <td>Sterile 1.5 ml tubes</td>
 <td>USA Scientific Inc</td>
 <td>1615-5500</td>
 <td></td>
 </tr>
 <tr>
 <td>10 ml borosilicate Pipets</td>
 <td>Fisher Scientific</td>
 <td>13-678-25E</td>
 <td></td>
 </tr>
 <tr>
 <td>Borosilicate culture tubes</td>
 <td>Fisher Scientific</td>
 <td>14-961-27</td>
 <td></td>
 </tr>
 <tr>
 <td>Uniskan I, ELISA plate reader</td>
 <td>Labsystem and Flow Laboratories, Helsinki, Finland</td>
 <td>Type 362</td>
 <td></td>
 </tr>
 </tbody>
</table>

a protocol for phage display and affinity selection using recombinant protein baits

Uso de fago recombinante como un andamio para presentar diversas porciones de proteínas codificadas por una biblioteca de ADNc clonado direccionalmente a las moléculas inmovilizadas de cebo es un medio eficaz para descubrir las interacciones. La técnica ha sido ampliamente utilizado para descubrir las interacciones proteína-proteína, pero la molécula cebo a ser cuestionada no tiene que ser restringida a las proteínas. El protocolo que aquí se presenta ha sido optimizado para permitir un modesto número de cebos que se proyectarán en réplicas de maximizar la identificación de clones independientes que presentan la misma proteína. Esto permite una mayor confianza en que interactúan las proteínas identificadas son interactores legítimos de la molécula de cebo. Control de la titulación del fago después de cada ronda de selección por afinidad proporciona información sobre cómo la selección por afinidad está progresando, así como sobre la eficacia de los controles negativos. Un medio de titulación de los fagos, y cómo y qué preparar con antelación para permitir que este proceso progrese lo más eficientementeposible, se presenta. Atributos de amplicones obtenidos después del aislamiento de la placa independiente se destacan que se puede utilizar para determinar lo bien que la selección por afinidad ha progresado. Se explican las técnicas de solución de problemas para minimizar los falsos positivos o de eludir persistentemente recuperó fago. Los medios para reducir la contaminación viral brote se discuten.

Ser capaz de poner en peligro la capacidad de la carnada para interactuar con las proteínas en fagos (metabólicamente envenenando el cebo) proporciona un control negativo potente para esta técnica. También es aconsejable para determinar si el cebo, cuando se une a la placa de microtitulación así, conserva su función. Ambos controles aumentará la confianza que interactúan, las proteínas en fagos recuperados por el cebo nonpoisoned son legítimas. Muestreo tres triplicado de cada poc...

Watch this Scientific Journal Video about A Protocol for Phage Display and Affinity Selection Using Recombinant Protein Baits at JoVE.com

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

La presentación en fagos es una técnica poderosa para capturar las proteínas o restos de proteínas que interactúan con una molécula inmovilizada de interés. Una vez que se ha tomado una decisión del tipo de fago biblioteca de cDNA para crear y pantalla, el protocolo aquí descrito permite la selección por afinidad eficaz que conduce a la identificación de los interactores.

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 ...

a-protocol-for-phage-display-affinity-selection-using-recombinant

Research

JoVE Journal

Biology

33.7K vistas.  University of Kentucky.  La presentación en fagos es una técnica poderosa para capturar las proteínas o restos de proteínas que interactúan con una molécula inmovilizada de interés. Una vez que se ha tomado una decisión del tipo de fago biblioteca de cDNA para crear y pantalla, el protocolo aquí descrito permite la selección por afinidad eficaz que conduce a la identificación de los interactores. 

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

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

CD1 proteins constitute a third class of antigen-presenting molecules. They are cell surface glycoproteins, expressed as approximately 50-kDa glycosylated heavy chains that are noncovalently associated with beta2-microglobulin. They bind lipids rather than peptides. Although their structure confirms the similarity of CD1 proteins to MHC class I and class II antigen presenting molecules, the mCD1d groove is relatively narrow, deep, and highly hydrophobic and it has two binding pockets instead of the several shallow pockets described for the classical MHC-encoded antigen-presenting molecules. Based upon their amino acid sequences, such a hydrobphobic groove provides an ideal environment for the binding of lipid antigens. The Natural Killer T (NKT) cells use their TCR to recognize glycolipids bound to or presented by CD1d. T cells reactive to lipids presented by CD1 have been involved in the protection against autoimmune and infectious diseases and in tumor rejection. Thus, the ability to identify, purify , and track the response of CD1-reactive NKT cell is of great importance . The generation of tetramers of alpha Galactosyl ceramide (a-Galcer) with CD1d has significant insight into the biology of NKT cells. Tetramers constructed from other CD1 molecules have also been generated and these new reagents have greatly expanded the knowledge of the functions of lipid-reactive T cells, with potential use in monitoring the response to lipid-based vaccines and in the diagnosis of autoimmune diseases and other treatments.

A Method to prepare Insect cells and infect them with baculovirus for the the purpose of production of recombinant mCD1d proteinand generating mCD1d tetramers.

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 ...

Investigación

Biología

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

Utilizando el sistema de fraccionamiento GELFREE 8100 de peso molecular basado en Fraccionamiento con la recuperación de la fase líquida

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Peptides from Phage Display Library Modulate Gene Expression in Mesenchymal Cells and Potentiate Osteogenesis in Unicortical Bone Defects

Two novel synthetic peptides accelerate bone formation and can be delivered using a collagen matrix. The aim of this study was to investigate the effects on bone repair in a unicortical defect model. Treatment of mesenchymal cells produced an increase in alkaline phosphatase activity, showed nodule formation by the cells, and increased the expression of genes for runx2, osterix, bone sialoprotein, and osteocalcin. A collagen sponge soaked with peptide promoted repair of bone defects, whereas the control was less effective. The results from this study demonstrated that mesenchymal cells treated with peptide in vitro differentiate towards osteogenesis, and, that peptides delivered in vivo using a collagen sponge promote the repair of unicortical defects.

A phage display library was used to identify peptide sequences that target bone. The objective was to investigate the effect of these peptides on mesenchymal cell differentiation and to determine their effect on bone regeneration.

Péptidos de la Biblioteca de Phage Display modular la expresión génica en las células mesenquimales y potenciar la osteogénesis en defectos óseos unicorticales

Two novel synthetic peptides accelerate bone formation and can be delivered using a collagen matrix. The aim of this study was to investigate the effects ...

Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification

In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction chromatography (HIC) commonly requires experimental determination (referred to as screening or "scouting") in order to select the most suitable chromatographic medium for purifying a given protein 1. The method presented here describes an automated approach to scouting for an optimal HIC media to be used in protein purification.
HIC separates proteins and other biomolecules from a crude lysate based on differences in hydrophobicity. Similar to affinity chromatography (AC) and ion exchange chromatography (IEX), HIC is capable of concentrating the protein of interest as it progresses through the chromatographic process. Proteins best suited for purification by HIC include those with hydrophobic surface regions and able to withstand exposure to salt concentrations in excess of 2 M ammonium sulfate ((NH4)2SO4). HIC is often chosen as a purification method for proteins lacking an affinity tag, and thus unsuitable for AC, and when IEX fails to provide adequate purification. Hydrophobic moieties on the protein surface temporarily bind to a nonpolar ligand coupled to an inert, immobile matrix. The interaction between protein and ligand are highly dependent on the salt concentration of the buffer flowing through the chromatography column, with high ionic concentrations strengthening the protein-ligand interaction and making the protein immobile (i.e. bound inside the column) 2. As salt concentrations decrease, the protein-ligand interaction dissipates, the protein again becomes mobile and elutes from the column. Several HIC media are commercially available in pre-packed columns, each containing one of several hydrophobic ligands (e.g. S-butyl, butyl, octyl, and phenyl) cross-linked at varying densities to agarose beads of a specific diameter 3. Automated column scouting allows for an efficient approach for determining which HIC media should be employed for future, more exhaustive optimization experiments and protein purification runs 4.
The specific protein being purified here is recombinant green fluorescent protein (GFP); however, the approach may be adapted for purifying other proteins with one or more hydrophobic surface regions. GFP serves as a useful model protein, due to its stability, unique light absorbance peak at 397 nm, and fluorescence when exposed to UV light 5. Bacterial lysate containing wild type GFP was prepared in a high-salt buffer, loaded into a Bio-Rad DuoFlow medium pressure liquid chromatography system, and adsorbed to HiTrap HIC columns containing different HIC media. The protein was eluted from the columns and analyzed by in-line and post-run detection methods. Buffer blending, dynamic sample loop injection, sequential column selection, multi-wavelength analysis, and split fraction eluate collection increased the functionality of the system and reproducibility of the experimental approach.

An automated method for identifying suitable hydrophobic interaction chromatography (HIC) media to be used in the process of protein purification is presented. The method utilizes a medium-pressure liquid chromatography system including automated buffer blending, dynamic sample loop injection, sequential column selection, multi-wavelength analysis, and split fraction eluate collection.

La interacción hidrofóbica automatizada cromatografía en columna de selección para uso en la purificación de proteínas

In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction ...

A Protocol for Computer-Based Protein Structure and Function Prediction

Genome sequencing projects have ciphered millions of protein sequence, which require knowledge of their structure and function to improve the understanding of their biological role. Although experimental methods can provide detailed information for a small fraction of these proteins, computational modeling is needed for the majority of protein molecules which are experimentally uncharacterized. The I-TASSER server is an on-line workbench for high-resolution modeling of protein structure and function. Given a protein sequence, a typical output from the I-TASSER server includes secondary structure prediction, predicted solvent accessibility of each residue, homologous template proteins detected by threading and structure alignments, up to five full-length tertiary structural models, and structure-based functional annotations for enzyme classification, Gene Ontology terms and protein-ligand binding sites. All the predictions are tagged with a confidence score which tells how accurate the predictions are without knowing the experimental data. To facilitate the special requests of end users, the server provides channels to accept user-specified inter-residue distance and contact maps to interactively change the I-TASSER modeling; it also allows users to specify any proteins as template, or to exclude any template proteins during the structure assembly simulations. The structural information could be collected by the users based on experimental evidences or biological insights with the purpose of improving the quality of I-TASSER predictions. The server was evaluated as the best programs for protein structure and function predictions in the recent community-wide CASP experiments. There are currently &gt;20,000 registered scientists from over 100 countries who are using the on-line I-TASSER server.

Guidelines for computer based structural and functional characterization of protein using the I-TASSER pipeline is described. Starting from query protein sequence, 3D models are generated using multiple threading alignments and iterative structural assembly simulations. Functional inferences are thereafter drawn based on matches to proteins with known structure and functions.

Un protocolo para la estructura de la proteína por computadora y la función de predicción

Genome sequencing projects have ciphered millions of protein sequence, which require knowledge of their structure and function to improve the ...

Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a demand for general methods that do not require target protein specific optimization1 . To achieve this, purification tags that genetically can be fused to the gene of interest are commonly used2 . The most widely used affinity handle is the hexa-histidine tag, which is suitable for purification under both native and denaturing conditions3 . The metabolic burden for producing the tag is low, but it does not provide as high specificity as competing affinity chromatography based strategies1,2.
Here, a bispecific purification tag with two different binding sites on a 46 amino acid, small protein domain has been developed. The albumin-binding domain is derived from Streptococcal protein G and has a strong inherent affinity to human serum albumin (HSA). Eleven surface-exposed amino acids, not involved in albumin-binding4 , were genetically randomized to produce a combinatorial library. The protein library with the novel randomly arranged binding surface (Figure 1) was expressed on phage particles to facilitate selection of binders by phage display technology. Through several rounds of biopanning against a dimeric Z-domain derived from Staphylococcal protein A5, a small, bispecific molecule with affinity for both HSA and the novel target was identified6 .
The novel protein domain, referred to as ABDz1, was evaluated as a purification tag for a selection of target proteins with different molecular weight, solubility and isoelectric point. Three target proteins were expressed in Escherishia coli with the novel tag fused to their N-termini and thereafter affinity purified. Initial purification on either a column with immobilized HSA or Z-domain resulted in relatively pure products. Two-step affinity purification with the bispecific tag resulted in substantial improvement of protein purity. Chromatographic media with the Z-domain immobilized, for example MabSelect SuRe, are readily available for purification of antibodies and HSA can easily be chemically coupled to media to provide the second matrix.
This method is especially advantageous when there is a high demand on purity of the recovered target protein. The bifunctionality of the tag allows two different chromatographic steps to be used while the metabolic burden on the expression host is limited due to the small size of the tag. It provides a competitive alternative to so called combinatorial tagging where multiple tags are used in combination1,7.

A novel and highly efficient two-step affinity chromatography protocol has been developed and is described in detail. The method is based on a small purification tag with two inherent affinities and is applicable to a wide range of target proteins with different properties.

Purificación de proteínas ortogonales Facilitado por un marcador de afinidad biespecíficos pequeños

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a ...

Identification of Protein Interacting Partners Using Tandem Affinity Purification

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein partners. There are many approaches that allow the identification of interacting partners, including the yeast two hybrid system, as well as pull down assays using recombinant proteins and immunoprecipitation of endogenous proteins followed by mass spectrometry identification1. Recent studies have highlighted the utility of double-affinity tag mediated purification, coupled with two specific elution steps in the identification of interacting proteins. This approach, termed Tandem Affinity Purification (TAP), was initially used in yeast2,3 but more recently has been adapted to use in mammalian cells4-8.
As proof-of-concept we have established a tandem affinity purification (TAP) method using the well-characterized eukaryotic translation initiation factor eIF4E9,10.The cellular translation factor eIF4E is a critical component of the cellular eIF4F complex involved in cap-dependent translation initiation10. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence8. To forgo the need for the generation of clonal cell lines, we developed a rapid system that relies on the expression of the TAP-tagged bait protein from an episomally maintained plasmid based on pMEP4 (Invitrogen). Expression of tagged murine eIF4E from this plasmid was controlled using the cadmium chloride inducible metallothionein promoter.
Lysis of the expressing cells and subsequent affinity purification via binding to rabbit IgG agarose, TEV protease cleavage, binding to streptavidin linked agarose and subsequent biotin elution identified numerous proteins apparently specific to the eIF4E pull-down (when compared to control cell lines expressing the TAP tag alone). The identities of the proteins were obtained by excision of the bands from 1D SDS-PAGE and subsequent tandem mass spectrometry. The identified components included the known eIF4E binding proteins eIF4G and 4EBP-1. In addition, other components of the eIF4F complex, of which eIF4E is a component were identified, namely eIF4A and Poly-A binding protein. The ability to identify not only known direct binding partners as well as secondary interacting proteins, further highlights the utility of this approach in the characterization of proteins of unknown function.

Tandem affinity purification is a robust approach for the identification of protein binding partners. As proof of concept, this methodology was applied to the well-characterized translation initiation factor eIF4E to co-precipitate the host cell factors involved in translation initiation. This method is easily adapted to any cellular or viral protein.

Identificación de los socios interacción de proteínas en tándem de purificación de afinidad

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein ...

A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein labeled, this method can be implemented for the investigation of protein-protein interactions. It is based on one hand on the recognition of the tagged protein by cobalt coated magnetic beads and on the other hand on the interaction between the tagged protein and a second specific protein that is labeled. First, the labeled and tagged proteins are mixed and incubated at room temperature. The magnetic beads, that recognize the tag, are added and the bound fraction of labeled protein is separated from the unbound fraction using magnets. The amount of labeled protein that is captured can be determined in an indirect way by measuring the signal of the labeled protein remained in the unbound fraction. The described liquid phase affinity assay is extremely useful when conformational conversion sensitive proteins are assayed. The development and application of the assay is demonstrated for the interaction between poliovirus and poliovirus recognizing nanobodies1. Since poliovirus is sensitive to conformational conversion2 when attached to a solid surface (unpublished results), the use of ELISA is limited and a liquid phase based system should therefore be preferred. An example of a liquid phase based system often used in polioresearch3,4 is the micro protein A-immunoprecipitation test5. Even though this test has proven its applicability, it requires an Fc-structure, which is absent in the nanobodies6,7. However, as another opportunity, these interesting and stable single-domain antibodies8 can be easily engineered with different tags. The widely used (His)6-tag shows affinity for bivalent ions such as nickel or cobalt, which can on their turn be easily coated on magnetic beads. We therefore developed this simple quantitative affinity capture assay based on cobalt coated magnetic beads. Poliovirus was labeled with 35S to enable unhindered interaction with the nanobodies and to make a quantitative detection feasible. The method is easy to perform and can be established with a low cost, which is further supported by the possibility of effectively regenerating the magnetic beads.

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. It is a reliable technique based on the interaction between magnetic beads and tagged proteins (e.g. nanobodies) on one hand and the affinity between the tagged protein and a second, labeled protein (e.g. poliovirus) on the other.

Una afinidad líquido fase de captura de ensayo utilizando partículas magnéticas para estudiar la interacción proteína-proteína: el ejemplo de poliomyelits-Nanobodies

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein ...

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in current research and a plethora of methods for their investigation is available1. Protein-protein interactions often are highly dynamic and may depend on subcellular localization, post-translational modifications and the local protein environment2. Therefore, they should be investigated in their natural environment, for which co-immunoprecipitation approaches are the method of choice3. Co-precipitated interaction partners are identified either by immunoblotting in a targeted approach, or by mass spectrometry (LC-MS/MS) in an untargeted way. The latter strategy often is adversely affected by a large number of false positive discoveries, mainly derived from the high sensitivity of modern mass spectrometers that confidently detect traces of unspecifically precipitating proteins. A recent approach to overcome this problem is based on the idea that reduced amounts of specific interaction partners will co-precipitate with a given target protein whose cellular concentration is reduced by RNAi, while the amounts of unspecifically precipitating proteins should be unaffected. This approach, termed QUICK for QUantitative Immunoprecipitation Combined with Knockdown4, employs Stable Isotope Labeling of Amino acids in Cell culture (SILAC)5 and MS to quantify the amounts of proteins immunoprecipitated from wild-type and knock-down strains. Proteins found in a 1:1 ratio can be considered as contaminants, those enriched in precipitates from the wild type as specific interaction partners of the target protein. Although innovative, QUICK bears some limitations: first, SILAC is cost-intensive and limited to organisms that ideally are auxotrophic for arginine and/or lysine. Moreover, when heavy arginine is fed, arginine-to-proline interconversion results in additional mass shifts for each proline in a peptide and slightly dilutes heavy with light arginine, which makes quantification more tedious and less accurate5,6. Second, QUICK requires that antibodies are titrated such that they do not become saturated with target protein in extracts from knock-down mutants.
Here we introduce a modified QUICK protocol which overcomes the abovementioned limitations of QUICK by replacing SILAC for 15N metabolic labeling and by replacing RNAi-mediated knock-down for affinity modulation of protein-protein interactions. We demonstrate the applicability of this protocol using the unicellular green alga Chlamydomonas reinhardtii as model organism and the chloroplast HSP70B chaperone as target protein7 (Figure 1). HSP70s are known to interact with specific co-chaperones and substrates only in the ADP state8. We exploit this property as a means to verify the specific interaction of HSP70B with its nucleotide exchange factor CGE19.

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

We present a variation of the QUICK (QUantitative Immunoprecipitation Combined with Knockdown) approach that was introduced previously to distinguish between true and false protein-protein interactions. Our approach is based on 15N metabolic labeling, the modulation of affinities of protein-protein interactions by the presence/absence of ATP, immunoprecipitation, and quantitative mass spectrometry.

El Protocolo para la identificación de las interacciones proteína-proteína base de<sup&gt; 15</sup&gt; N metabólico Etiquetado, inmunoprecipitación, cuantitativa Espectrometría de Masas y Modulación Affinity

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in ...

High-throughput Purification of Affinity-tagged Recombinant Proteins

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further biochemical analyses to obtain detailed insights into structure/function relationships. Advances in oligonucleotide- and gene synthesis technology make large-scale mutagenesis strategies increasingly feasible, including the substitution of target residues by all 19 other amino acids. Gain- or loss-of-function phenotypes then allow systematic conclusions to be drawn, such as the contribution of particular residues to catalytic activity, protein stability and/or protein-protein interaction specificity.
In order to attribute the different phenotypes to the nature of the mutation - rather than to fluctuating experimental conditions - it is vital to purify and analyse the proteins in a controlled and reproducible manner. High-throughput strategies and the automation of manual protocols on robotic liquid-handling platforms have created opportunities to perform such complex molecular biological procedures with little human intervention and minimal error rates1-5.
Here, we present a general method for the purification of His-tagged recombinant proteins in a high-throughput manner. In a recent study, we applied this method to a detailed structure-function investigation of TFIIB, a component of the basal transcription machinery. TFIIB is indispensable for promoter-directed transcription in vitro and is essential for the recruitment of RNA polymerase into a preinitiation complex6-8. TFIIB contains a flexible linker domain that penetrates the active site cleft of RNA polymerase9-11. This linker domain confers two biochemically quantifiable activities on TFIIB, namely (i) the stimulation of the catalytic activity during the 'abortive' stage of transcript initiation, and (ii) an additional contribution to the specific recruitment of RNA polymerase into the preinitiation complex4,5,12 . We exploited the high-throughput purification method to generate single, double and triple substitution and deletions mutations within the TFIIB linker and to subsequently analyse them in functional assays for their stimulation effect on the catalytic activity of RNA polymerase4. Altogether, we generated, purified and analysed 381 mutants - a task which would have been time-consuming and laborious to perform manually. We produced and assayed the proteins in multiplicates which allowed us to appreciate any experimental variations and gave us a clear idea of the reproducibility of our results.
This method serves as a generic protocol for the purification of His-tagged proteins and has been successfully used to purify other recombinant proteins. It is currently optimised for the purification of 24 proteins but can be adapted to purify up to 96 proteins.

We describe a method for the affinity-tagged purification of recombinant proteins using liquid-handling robotics. This method is generally applicable to the small-scale purification of soluble His-tagged proteins in a high-throughput format.

De alto rendimiento de purificación de afinidad de proteínas marcadas con recombinantes

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further ...