Ce travail a été soutenu financièrement par les subventions K22AI099165 (AWB), P41GM103390 (KWM) et P01GM107012 (KWM) des Instituts nationaux de la santé, et par des fonds provenant du Roy J. Carver Département de biochimie, biophysique et biologie moléculaire à l&#39;Université de l&#39;Iowa . Le contenu de ce travail est de la seule responsabilité des auteurs et ne représentent pas nécessairement les vues officielles de la NIH.

All authors declare no competing financial interests in this manuscript.

Ce protocole illustre l&#39;expression de la protéine par l&#39;intermédiaire de la transfection transitoire de cellules HEK293F ou S. Les conditions de transfection optimales établies dans les laboratoires et Barb Moremen emploient une combinaison critique de concentrations de densité de cellules et de réactifs pour réaliser la transfection à haut rendement. Considérations critiques lors de la mise en œuvre de ce protocole comprennent: le maintien d&#39;une culture stable avant la transfection (avec la culture...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/53568">High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension</a>. The Authors section was updated. from:
Ganesh P. Subedi1 
Roy W. Johnson2 
Heather A. Moniz2 
Kelley W. Moremen2 
Adam Barb1 
1The Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University 
2Complex Carbohydrate Research Center, University of Georgia
to
Ganesh P. Subedi1 
Roy W. Johnson2 
Heather A. Moniz2 
Kelley W. Moremen2 
Adam W. Barb1 
1The Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University 
2Complex Carbohydrate Research Center, University of Georgia

An erratum was issued for:&#160;<a href="https://www.jove.com/t/53568">High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension</a>. The Authors section was updated.

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

Produire des rendements élevés de protéines humaines pliées de manière appropriée et modifications post-traductionnelles pour l&#39;analyse détaillée de la structure et de la fonction demeure un défi important. Un grand nombre de systèmes d&#39;expression sont disponibles qui produisent des protéines recombinantes avec fonction native-like et le comportement. Systèmes d&#39;expression bactériens, principalement des souches d&#39;Escherichia coli, représentent les outils les plus accessibles et couramment utilisés dans le domaine de la recherche, en raison de la simplicité de ces systèmes d&#39;expression, bien que la levure, des plantes, les insectes et les mammifères sont également décrits 1-4. Cependant, la plupart de ces systèmes sont incapables de modification post-traductionnelle appropriée des protéines cibles. Un intérêt fondamental des laboratoires Barb et Moremen est la production de protéines eucaryotes avec une glycosylation appropriée. Beaucoup de protéines humaines exigent glycosylation appropriée pour le bon fonctionnement (voir 5). Le eucaryotemachines de glycosylation est vaste et capable de faire un large éventail de modifications, incluant à la fois l&#39;asparagine (N) - et de la sérine / thréonine (O) lié en glycanes complexes 6. On estime que&gt; 50% des protéines humaines sont N-glycosylées 7. Glycanes sont des composantes essentielles de nombreuses protéines, y compris des anticorps thérapeutiques monoclonaux, l&#39;érythropoïétine, et les facteurs de coagulation du sang comme le facteur IX, pour ne nommer que quelques-uns. Bien que plusieurs méthodes existent pour préparer des protéines convenablement N-glycosylée et vont de purement synthétique 8-10, 11-14 à chimioenzymatiques ou la récupération de systèmes recombinants d&#39;ingénierie 15-20, sans surprise, des systèmes d&#39;expression humains ont jusqu&#39;ici avéré être le plus robuste des procédés pour générer des protéines humaines. De nombreuses glycoprotéines humaines thérapeutiques sont produits dans des systèmes recombinants en utilisant des cellules de mammifères. Systèmes de la note sont l&#39;ovaire de hamster chinois (CHO), myélome de souris (NS0), Baby Hamster Kidney (BHK), de rein embryonnaire humain (HEK-293) et des lignées de cellules rétiniennes humaines qui sont utilisés dans l&#39;adhésion ou de la suspension de culture pour la production de protéine 4,21,22. Cependant, les systèmes d&#39;expression de protéines de mammifère ont nécessité la production de lignées cellulaires stables, des milieux de croissance et le substrat coûteuses procédures de transfection assistée 23. La transfection de cellules de mammifère est obtenue à l&#39;aide de nombreux agents, y compris les phosphates de calcium, les polymères cationiques 24,25 (DEAE-dextrane, le polybrène, la polylysine, polyéthylimine (PEI)) ou des liposomes cationiques chargés positivement 26-29. PEI est un polycationique, chargé, linéaire ou ramifié en polymère (25 kDa) 26 qui forme un complexe stable avec l&#39;ADN et est endocytose. Lors de l&#39;acidification de l&#39;endosome, PEI est pensé pour gonfler, ce qui conduit à la rupture des endosomes et la libération de l&#39;ADN dans le cytoplasme de 26,30. Jusqu&#39;à récemment, la transfection transitoire dans suspensila culture a été réalisée par la formation du complexe ADN / PEI avant suivie de l&#39;addition à la culture de cellules 29. Cependant, Würm et ses collaborateurs ont rapporté un protocole est performant optimisé pour une production de protéines recombinantes dans des cellules HEK293 qui ont formé un complexe ADN / PEI in situ 31,32. Ceci évite préparation, la stérilisation du complexe, et l&#39;échange de tampon dans un milieu de culture. Poursuite de l&#39;optimisation en incluant des plasmides d&#39;expression amélioration conduit à une augmentation significative de rendement 33. Est ici une méthode qui se fonde sur ces avancées et est largement applicable. Conditions d&#39;expression peuvent aussi être modifiées pour influer sur la composition de N-glycanes. La lignée cellulaire HEK293S, avec une deletion de gène qui interrompt le traitement de N-glycane à un stade intermédiaire, conduit à l&#39;expression de protéines avec des uniformes N-glycanes comprenant 2 résidus N-acétylglucosamine et cinq résidus de mannose (Man 5 GlcNAc 2) 34, 35. Ces cellulespas la N-acétylglucosaminyl transférase I (GntI) gène qui est nécessaire pour la N-glycane en aval traitement 36,37. L&#39;utilisation d&#39;inhibiteurs de glycosyltransférase comprenant kifunensine, des analogues d&#39;acide sialique et l&#39;analogue de fucose et de 2-désoxy-2-fluoro-fucose a des effets de traitement et les limites de N-glycane 38-41 similaires. Le protocole indiqué ici utilise le vecteur pGEN2 comme représenté sur la figure 1 42,43, PEI transfection transitoire dans des cellules assistée lignes de mammifères ou des cellules HEK293F (HEK293S), et la récupération des rendements élevés de protéine glycosylée de façon appropriée. Ce système est robuste et peut accueillir divers facteurs, notamment marquage isotopique et de l&#39;ingénierie glycane pour la production de grandes titres de protéines recombinantes.

<table>
	<tbody>
		<tr>
			<td>Biosafety cabinet&#160;</td>
			<td>NuAire, Inc.</td>
			<td>CellGard ES NU-S475-400</td>
			<td>Class II, Type A2 Biological Safety Cabinet</td>
		</tr>
		<tr>
			<td>Incubation shaker</td>
			<td>INFORS HT</td>
			<td></td>
			<td>Multitron Cell&#160;</td>
		</tr>
		<tr>
			<td>Medium A: FreeStyle Expression Medium&#160;</td>
			<td>Life Technologies</td>
			<td>12338-018</td>
			<td></td>
		</tr>
		<tr>
			<td>Medium B: ExCell 293 Serum-Free Medium&#160;</td>
			<td>SIGMA</td>
			<td>14571C&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>125 Erlenmeyer Flask with Vented Cap</td>
			<td>Corning Incorporated/Life Sciences</td>
			<td>431143</td>
			<td></td>
		</tr>
		<tr>
			<td>250 Erlenmeyer Flask with Vented Cap</td>
			<td>Corning Incorporated/Life Sciences</td>
			<td>431144</td>
			<td></td>
		</tr>
		<tr>
			<td>FreeStyle HEK 293F Cells</td>
			<td>Life Technologies</td>
			<td>R790-07</td>
			<td></td>
		</tr>
		<tr>
			<td>1 ml Disposable serological pipette</td>
			<td>Fisher Scietific</td>
			<td>13-676-10B</td>
			<td></td>
		</tr>
		<tr>
			<td>10 ml Disposable serological pipette</td>
			<td>Fisher Scietific</td>
			<td>13-676-10J</td>
			<td></td>
		</tr>
		<tr>
			<td>25 ml Disposable serological pipette</td>
			<td>Fisher Scietific</td>
			<td>13-676-10K</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettor (Pipet-Aid XP)</td>
			<td>Drummond Scientific</td>
			<td>161263</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue Solution&#160;</td>
			<td>Thermo Scientific&#160;</td>
			<td>SV30084.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Counting slides&#160;</td>
			<td>Bio-Rad</td>
			<td>145-0011</td>
			<td></td>
		</tr>
		<tr>
			<td>TC20 Automated Cell Counter&#160;</td>
			<td>Bio-Rad&#160;</td>
			<td>145-0102</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylenimine (PEI)</td>
			<td>Polysciences Inc.</td>
			<td>23966</td>
			<td>Prepare stock solution at a concentration of 1 mg/ml in a buffer containing 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and 150 mM NaCl (pH 7.5). Dissolve PEI completely; sterilize through 0.22 &#956;M syringe filter and store at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)</td>
			<td>EMD Chemicals Inc.</td>
			<td>7365-45-9</td>
			<td></td>
		</tr>
		<tr>
			<td>25 mm Syringe Filter, 0.22 &#956;M</td>
			<td>Fisher Scietific</td>
			<td>09-719A</td>
			<td></td>
		</tr>
		<tr>
			<td>Trisaminomethane (Tris base)</td>
			<td>Fisher Scietific</td>
			<td>BP152-1</td>
			<td></td>
		</tr>
		<tr>
			<td>XL1-Blue</td>
			<td>Stratagene</td>
			<td>222249</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypton</td>
			<td>Fisher Scietific</td>
			<td>BP1421-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Fluka Analytical&#160;</td>
			<td>92144</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>BDH chemicals</td>
			<td>BDH8014</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmid Purification Kit</td>
			<td>QIAGEN</td>
			<td>12145</td>
			<td></td>
		</tr>
		<tr>
			<td>Valproic (VPA)</td>
			<td>SIGMA</td>
			<td>P4543</td>
			<td>Prepare stock solution of 220 mM&#160; in water, sterilize by passage through a sterile 0.22 mm filter and store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Corning&#63720; 250 mL Centrifuge Tube&#160;</td>
			<td>Corning Incorporated/Life Sciences</td>
			<td>430776</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Thermo Scientific&#160;</td>
			<td>EW-17707-65</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein A-Sepharose column</td>
			<td>SIGMA</td>
			<td>P9424</td>
			<td></td>
		</tr>
		<tr>
			<td>Ni-NTA superflow</td>
			<td>QIAGEN</td>
			<td>30430</td>
			<td></td>
		</tr>
		<tr>
			<td>3-(N-morpholino)propanesulfonic acid (MOPS)</td>
			<td>Fisher Scietific</td>
			<td>BP308-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Fisher Scietific</td>
			<td>BP381-500</td>
			<td></td>
		</tr>
		<tr>
			<td>10 kDa molecular weight cut-off Amicon&#174; Ultra centrifugal filters&#160;</td>
			<td>Millipore</td>
			<td>UFC901096</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate</td>
			<td>ALDRICH</td>
			<td>L3771</td>
			<td></td>
		</tr>
		<tr>
			<td>Beta-mercaptoethanol</td>
			<td>ALDRICH</td>
			<td>M6250</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>SIGMA</td>
			<td>G5516</td>
			<td></td>
		</tr>
		<tr>
			<td>Precision Plus Protein All Blue Standards</td>
			<td>Bio-Rad</td>
			<td>161-0373</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic Acid, Glacial&#160;</td>
			<td>Fisher Scietific</td>
			<td>64-19-7</td>
			<td></td>
		</tr>
		<tr>
			<td>coomassie brilliant blue&#160;</td>
			<td>Bio-Rad&#160;</td>
			<td>161-0406</td>
			<td></td>
		</tr>
		<tr>
			<td>MALDI-TOFMS Voyager-DE PRO&#160;</td>
			<td>Applied Biosystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>15N labeled L-Tyrosine</td>
			<td>ALDRICH</td>
			<td>332151</td>
			<td></td>
		</tr>
		<tr>
			<td>15N labeled L-Lysine</td>
			<td>ALDRICH</td>
			<td>592900</td>
			<td></td>
		</tr>
		<tr>
			<td>Unlabeled L-Phenylalanine</td>
			<td>SIGMA-ALDRICH</td>
			<td>P2126</td>
			<td></td>
		</tr>
		<tr>
			<td>13C6-Glucose</td>
			<td>ALDRICH</td>
			<td>389374</td>
			<td></td>
		</tr>
		<tr>
			<td>2-deoxy-2-fluoro-l-fucose&#160;</td>
			<td>SANTA CRUZ Biotechnology</td>
			<td>sc-283123</td>
			<td></td>
		</tr>
	</tbody>
</table>

high yield expression of recombinant human proteins with the transient transfection of hek293 cells in suspension

L&#39;art de la production de protéines recombinantes avec des modifications post-traductionnelles complexes représente un défi majeur pour les études de structure et la fonction. La mise en place rapide et de récupération élevés de lignées cellulaires de mammifères transitoirement transfectées aborde cette barrière et est un moyen efficace d&#39;exprimer des protéines qui sont naturellement acheminée par le ER et Golgi voie de sécrétion médiée. Voici un protocole pour l&#39;expression de protéine en utilisant la HEK293F humain et des lignées de cellules transfectées HEK293S avec un vecteur d&#39;expression de mammifère destiné à des rendements élevés de protéines. L&#39;applicabilité de ce système est démontrée en utilisant trois glycoprotéines représentatives qui ont exprimé avec des rendements entre 95-120 mg de protéine purifiée récupérée par litre de culture. Ces protéines sont le FcyRIIIa humain et la sialyltransférase α2-6 de rat, ST6GalI, tous deux exprimés avec une fusion GFP N-terminale, ainsi que l&#39;immunoglobuline humaine non modifiée G1 Fc. Cette robustes uti systèmeLizes un milieu exempt de sérum qui peut être adapté pour l&#39;expression de protéines et d&#39;hydrates de carbone pour des études structurales par spectrométrie de masse et spectroscopie de résonance magnétique nucléaire isotopiquement enrichis. En outre, la composition du N-glycane peut être réglée par addition d&#39;une petite molécule pour empêcher certaines modifications glycanniques d&#39;une manière qui ne réduit pas le rendement.

Expression de la protéine de haut niveau et de la pureté Ce système d&#39;expression optimisé généré un rendement élevé de protéines glycosylées. Un motif typique est montrée dans l&#39;expression de Fc-IgG1 (figure 1). Dans ce cas, jour 0 est le jour de la transfection suivie par Jour 1 (dilution) et les jours de culture subséquentes jusqu&#39;à Jour 5. expression de la protéine est analysée e...

Watch this Scientific Journal Video about High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension at JoVE.com

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

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 expression de protéines recombinantes humaines avec la transfection transitoire de cellules HEK293 en suspension

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

high-yield-expression-recombinant-human-proteins-with-transient

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JoVE Journal

Biology

29.5K vues.  Iowa State University. 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.

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

Production of Replication-Defective Retrovirus by Transient Transfection of 293T cells

Our lab studies human myeloproliferative diseases induced by such oncogenes as Bcr-Abl or growth factor receptor-derived oncogenes (ZNF198-FGFR1, Bcr-PDGFR&#945;, etc.). We are able to model and study a human-like disease in our mouse model, by transplanting bone marrow cells previously infected with a retrovirus expressing the oncogene of interest. Replication-defective retrovirus encoding a human oncogene and a marker (GFP, RFP, antibiotic resistance gene, etc.) is produced by a transient transfection protocol using 293T cells, a human renal epithelial cell line transformed by the adenovirus E1A gene product. 293 cells have the unusual property of being highly transfectable by calcium phosphate (CaPO4), with up to 50-80% transfection efficiency readily attainable. Here, we co-transfect 293 cells with a retroviral vector expressing the oncogene of interest and a plasmid that expresses the gag-pol-env packaging functions, such as the single-genome packaging constructs kat or pCL, in this case the EcoPak plasmid. The initial transfection is further improved by use of chloroquine. Stocks of ecotropic virus, collected as culture supernatant 48 hrs. post-transfection, can be stored at -80&deg;C and used for infection of cell-lines in view of transformation and in vitro studies, or primary cells such as mouse bone marrow cells, that can then be used for transplant in our mouse model.

This technique demonstrates an efficient way to prepare replication-defective retroviral stocks encoding a human oncogene, and subsequently used for induction of myeloproliferative disease in the mouse model.

Production de réplication défectueuse Rétrovirus par transfection transitoire de cellules 293T

Our lab studies human myeloproliferative diseases induced by such oncogenes as Bcr-Abl or growth factor receptor-derived oncogenes (ZNF198-FGFR1, ...

Recherche

Biologie

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.

Coloration des protéines dans les gels de Coomassie G-250, sans solvant et l&#39;acide acétique organique

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.

Utilisation du système d&#39;électroporation Gene Pulser MXcell à transfecter des cellules primaires à haut rendement

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

Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris

Protein expression in the microbial eukaryotic host Pichia pastoris offers the possibility to generate high amounts of recombinant protein in a fast and easy to use expression system.
As a single-celled microorganism P. pastoris is easy to manipulate and grows rapidly on inexpensive media at high cell densities. Being a eukaryote, P. pastoris is able to perform many of the post-translational modifications performed by higher eukaryotic cells and the obtained recombinant proteins undergo protein folding, proteolytic processing, disulfide bond formation and glycosylation [1].
As a methylotrophic yeast P. pastoris is capable of metabolizing methanol as its sole carbon source. The strong promoter for alcohol oxidase, AOX1, is tightly regulated and induced by methanol and it is used for the expression of the gene of interest. Accordingly, the expression of the foreign protein can be induced by adding methanol to the growth medium [2; 3].
Another important advantage is the secretion of the recombinant protein into the growth medium, using a signal sequence to target the foreign protein to the secretory pathway of P. pastoris. With only low levels of endogenous protein secreted to the media by the yeast itself and no added proteins to the media, a heterologous protein builds the majority of the total protein in the medium and facilitates following protein purification steps [3; 4].
The vector used here (pPICZ&alpha;A) contains the AOX1 promoter for tightly regulated, methanol-induced expression of the gene of interest; the &alpha;-factor secretion signal for secretion of the recombinant protein, a Zeocin resistance gene for selection in both E. coli and Pichia and a C-terminal peptide containing the c-myc epitope and a polyhistidine (6xHis) tag for detection and purification of a recombinant protein. We also show western blot analysis of the recombinant protein using the specific Anti-myc-HRP antibody recognizing the c-myc epitope on the parent vector.

Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris

The protocol describes protein expression using the methylotrophic yeast Pichia pastoris. The preparation of electrocompetent yeast cells, transformation of the vector with the gene of interest into P. pastoris and yeast DNA purification are also performed. Western blot analysis and protein purification build the last steps in this protein expression protocol.

Expression de protéines recombinantes dans le levure méthylotrophe<emPichia pastoris></em

Protein expression in the microbial eukaryotic host Pichia pastoris offers the possibility to generate high amounts of recombinant protein in a fast and ...

Optimized Transfection Strategy for Expression and Electrophysiological Recording of Recombinant Voltage-Gated Ion Channels in HEK-293T Cells

The in vitro expression and electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T) is a ubiquitous research strategy. HEK-293T cells must be plated onto glass coverslips at low enough density so that they are not in contact with each other in order to allow for electrophysiological recording without confounding effects due to contact with adjacent cells. Transfected channels must also express with high efficiency at the plasma membrane for whole-cell patch clamp recording of detectable currents above noise levels. Heterologous ion channels often require long incubation periods at 28&deg;C after transfection in order to achieve adequate membrane expression, but there are increasing losses of cell-coverslip adhesion and membrane stability at this temperature. To circumvent this problem, we developed an optimized strategy to transfect and plate HEK-293T cells. This method requires that cells be transfected at a relatively high confluency, and incubated at 28&deg;C for varying incubation periods post-transfection to allow for adequate ion channel protein expression. Transfected cells are then plated onto glass coverslips and incubated at 37&deg;C for several hours, which allows for rigid cell attachment to the coverslips and membrane restabilization. Cells can be recorded shortly after plating, or can be transferred to 28&deg;C for further incubation. We find that the initial incubation at 28&deg;C, after transfection but before plating, is key for the efficient expression of heterologous ion channels that normally do not express well at the plasma membrane. Positively transfected, cultured cells are identified by co-expressed eGFP or eGFP expressed from a bicistronic vector (e.g. pIRES2-EGFP) containing the recombinant ion channel cDNA just upstream of an internal ribosome entry site and an eGFP coding sequence. Whole-cell patch clamp recording requires specialized equipment, plus the crafting of polished recording electrodes and L-shaped ground electrodes from borosilicate glass. Drug delivery to study the pharmacology of ion channels can be achieved by directly micropipetting drugs into the recording dish, or by using microperfusion or gravity flow systems that produce uninterrupted streams of drug solution over recorded cells.

Reliable method for highly efficient in vitro expression and subsequent electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T).

Stratégie de transfection optimisés pour l&#39;expression et l&#39;enregistrement électrophysiologique des recombinants canaux ioniques voltage-dépendants présents au HEK-293T

The in vitro expression and electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T) ...

Absolute Quantum Yield Measurement of Powder Samples

Measurement of fluorescence quantum yield has become an important tool in the search for new solutions in the development, evaluation, quality control and research of illumination, AV equipment, organic EL material, films, filters and fluorescent probes for bio-industry.
Quantum yield is calculated as the ratio of the number of photons absorbed, to the number of photons emitted by a material. The higher the quantum yield, the better the efficiency of the fluorescent material.
For the measurements featured in this video, we will use the Hitachi F-7000 fluorescence spectrophotometer equipped with the Quantum Yield measuring accessory and Report Generator program. All the information provided applies to this system. 
Measurement of quantum yield in powder samples is performed following these steps:
<ol>
 <li>Generation of instrument correction factors for the excitation and emission monochromators. This is an important requirement for the correct measurement of quantum yield. It has been performed in advance for the full measurement range of the instrument and will not be shown in this video due to time limitations.</li>
 <li>Measurement of integrating sphere correction factors. The purpose of this step is to take into consideration reflectivity characteristics of the integrating sphere used for the measurements.</li>
 <li>Reference and Sample measurement using direct excitation and indirect excitation.</li>
 <li>Quantum Yield calculation using Direct and Indirect excitation. Direct excitation is when the sample is facing directly the excitation beam, which would be the normal measurement setup. However, because we use an integrating sphere, a portion of the emitted photons resulting from the sample fluorescence are reflected by the integrating sphere and will re-excite the sample, so we need to take into consideration indirect excitation. This is accomplished by measuring the sample placed in the port facing the emission monochromator, calculating indirect quantum yield and correcting the direct quantum yield calculation.</li>
 <li>Corrected quantum yield calculation.</li>
 <li>Chromaticity coordinates calculation using Report Generator program.</li>
</ol>
The Hitachi F-7000 Quantum Yield Measurement System offer advantages for this
application, as follows:
<ul>
 <li>High sensitivity (S/N ratio 800 or better RMS). Signal is the Raman band of water measured under the following conditions: Ex wavelength 350 nm, band pass Ex and Em 5 nm, response 2 sec), noise is measured at the maximum of the Raman peak. High sensitivity allows measurement of samples even with low quantum yield. Using this system we have measured quantum yields as low as 0.1 for a sample of salicylic acid and as high as 0.8 for a sample of magnesium tungstate.</li>
 <li>Highly accurate measurement with a dynamic range of 6 orders of magnitude allows for measurements of both sharp scattering peaks with high intensity, as well as broad fluorescence peaks of low intensity under the same conditions. </li>
 <li>High measuring throughput and reduced light exposure to the sample, due to a high scanning speed of up to 60,000 nm/minute and automatic shutter function. </li>
 <li>Measurement of quantum yield over a wide wavelength range from 240 to 800 nm.</li>
 <li>Accurate quantum yield measurements are the result of collecting instrument spectral response and integrating sphere correction factors before measuring the sample.</li>
 <li>Large selection of calculated parameters provided by dedicated and easy to use software.
</li>
</ul> 
During this video we will measure sodium salicylate in powder form which is known to have a quantum yield value of 0.4 to 0.5.

In this video we will demonstrate measuring and calculating absolute quantum yield and chromaticity coordinates directly in powder samples using the Hitachi F-7000 Quantum Yield Measuring System.

Absolute mesure du rendement quantique des échantillons de poudre

Measurement of fluorescence quantum yield has become an important tool in the search for new solutions in the development, evaluation, quality control and ...

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

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. However, prokaryotic hosts are often not as appropriate for expression of human, viral or eukaryotic proteins due to toxicity of the foreign macromolecule, differences in the protein folding machinery, or due to the lack of particular co- or post-translational modifications in bacteria. Expression systems based on yeast (P. pastoris or S. cerevisiae) 1,2, baculovirus-infected insect (S. frugiperda or T. ni) cells 3, and cell-free in vitro translation systems 2,4 have been successfully used to produce mammalian proteins. Intuitively, the best match is to use a mammalian host to ensure the production of recombinant proteins that contain the proper post-translational modifications. A number of mammalian cell lines (Human Embryonic Kidney (HEK) 293, CV-1 cells in Origin carrying the SV40 larget T-antigen (COS), Chinese Hamster Ovary (CHO), and others) have been successfully utilized to overexpress milligram quantities of a number of human proteins 5-9. However, the advantages of using mammalian cells are often countered by higher costs, requirement of specialized laboratory equipment, lower protein yields, and lengthy times to develop stable expression cell lines. Increasing yield and producing proteins faster, while keeping costs low, are major factors for many academic and commercial laboratories.
Here, we describe a time- and cost-efficient, two-part procedure for the expression of secreted human proteins from adherent HEK 293T cells. This system is capable of producing microgram to milligram quantities of functional protein for structural, biophysical and biochemical studies. The first part, multiple constructs of the gene of interest are produced in parallel and transiently transfected into adherent HEK 293T cells in small scale. The detection and analysis of recombinant protein secreted into the cell culture medium is performed by western blot analysis using commercially available antibodies directed against a vector-encoded protein purification tag. Subsequently, suitable constructs for large-scale protein production are transiently transfected using polyethyleneimine (PEI) in 10-layer cell factories. Proteins secreted into litre-volumes of conditioned medium are concentrated into manageable amounts using tangential flow filtration, followed by purification by anti-HA affinity chromatography. The utility of this platform is proven by its ability to express milligram quantities of cytokines, cytokine receptors, cell surface receptors, intrinsic restriction factors, and viral glycoproteins. This method was also successfully used in the structural determination of the trimeric ebolavirus glycoprotein 5,10.
In conclusion, this platform offers ease of use, speed and scalability while maximizing protein quality and functionality. Moreover, no additional equipment, other than a standard humidified CO2 incubator, is required. This procedure may be rapidly expanded to systems of greater complexity, such as co-expression of protein complexes, antigens and antibodies, production of virus-like particles for vaccines, or production of adenoviruses or lentiviruses for transduction of difficult cell lines.

In the post-human genomics era, the availability of recombinant proteins in native conformations is crucial to structural, functional and therapeutic research and development. Here, we describe a test- and large-scale protein expression system in human embryonic kidney 293T cells that can be used to produce a variety of recombinant proteins.

Une plate-forme d&#39;expression pratique et général pour la production de protéines sécrétées à partir de cellules humaines

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

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.

À haut débit Affinity Purification de protéines recombinantes-étiquette

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 ...

Efficient Agroinfiltration of Plants for High-level Transient Expression of Recombinant Proteins

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing worldwide demand for pharmaceuticals due to its limited scalability and high cost. Plants have shown to be one of the most promising alternative pharmaceutical production platforms that are robust, scalable, low-cost and safe. The recent development of virus-based vectors has allowed rapid and high-level transient expression of recombinant proteins in plants. To further optimize the utility of the transient expression system, we demonstrate a simple, efficient and scalable methodology to introduce target-gene containing Agrobacterium into plant tissue in this study. Our results indicate that agroinfiltration with both syringe and vacuum methods have resulted in the efficient introduction of Agrobacterium into leaves and robust production of two fluorescent proteins; GFP and DsRed. Furthermore, we demonstrate the unique advantages offered by both methods. Syringe infiltration is simple and does not need expensive equipment. It also allows the flexibility to either infiltrate the entire leave with one target gene, or to introduce genes of multiple targets on one leaf. Thus, it can be used for laboratory scale expression of recombinant proteins as well as for comparing different proteins or vectors for yield or expression kinetics. The simplicity of syringe infiltration also suggests its utility in high school and college education for the subject of biotechnology. In contrast, vacuum infiltration is more robust and can be scaled-up for commercial manufacture of pharmaceutical proteins. It also offers the advantage of being able to agroinfiltrate plant species that are not amenable for syringe infiltration such as lettuce and Arabidopsis. Overall, the combination of syringe and vacuum agroinfiltration provides researchers and educators a simple, efficient, and robust methodology for transient protein expression. It will greatly facilitate the development of pharmaceutical proteins and promote science education.

Plants offer a novel system for the production of pharmaceutical proteins on a commercial scale that is more scalable, cost-efficient and safe than current expression paradigms. In this study, we report a simple and convenient, yet scalable approach to introduce target-gene containing Agrobacterium tumefaciens into plants for protein transient expression.

Agroinfiltration efficace des plantes pour l&#39;expression transitoire de haut niveau de protéines recombinantes

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing ...

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. However, purifying proteins is sometimes challenging since many proteins are expressed in an insoluble form. When working with difficult or multiple targets it is therefore recommended to use high throughput (HTP) protein expression screening on a small scale (1-4&#160;ml cultures) to quickly identify conditions for soluble expression. To cope with the various structural genomics programs of the lab, a quantitative (within a range of 0.1-100 mg/L culture of recombinant protein) and HTP protein expression screening protocol was implemented and validated on thousands of proteins. The protocols were automated with the use of a liquid handling robot but can also be performed manually without specialized equipment.
Disulfide-rich venom proteins are gaining increasing recognition for their potential as therapeutic drug leads. They can be highly potent and selective, but their complex disulfide bond networks make them challenging to produce. As a member of the FP7 European Venomics project (<a href="http://www.venomics.eu">www.venomics.eu</a>), our challenge is to develop successful production strategies with the aim of producing thousands of novel venom proteins for functional characterization. Aided by the redox properties of disulfide bond isomerase DsbC, we adapted our HTP production pipeline for the expression of oxidized, functional venom peptides in the E. coli cytoplasm. The protocols are also applicable to the production of diverse disulfide-rich proteins. Here we demonstrate our pipeline applied to the production of animal venom proteins. With the protocols described herein it is likely that soluble disulfide-rich proteins will be obtained in as little as a week. Even from a small scale, there is the potential to use the purified proteins for validating the oxidation state by mass spectrometry, for characterization in pilot studies, or for sensitive micro-assays.

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

A protocol for the quantitative, high throughput expression screening and analytical purification of fusion proteins from small-scale Escherichia coli cultures is described and applied to the expression of disulfide-rich animal venom protein targets.

High Throughput Screening Expression quantitative et purification appliquée à recombinant disulfure riches protéines venin produit dans<em&gt; E. coli</em

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. ...