Name: a method for production of recombinant mcd1d protein in insect cells.
Uploaded: 2007-12-10T22:00:00
Description: Watch this Scientific Journal Video about A Method For Production of Recombinant mCD1d Protein in Insect Cells. at JoVE.com

<p class="jove_step">I. Thaw the Cells</p>
<p class="jove_content">Take the frozen vial of TN5 cells, and thaw it in a 37&deg;C water bath. Add 5ml of insect Express medium (Invitrogen, catalog number-10486) in 25cm<sup>2 </sup>T.C flask. Note that Express five comes without Glutamine; need to add 10ml of 100X glutamine pen strep or Glutamine alone to 1 lt media. Add TN5 cells into it and incubate for 30 minutes in a 27&deg;C incubator. Then, aspirate the medium with DMSO gently without disturbing the cells attached to the bottom of flask. Add ...

<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> <em style='font-style: italic'>J. Exp. Med</em>. <b>191</b>, 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> <em style='font-style: italic'>Nature</em>. <b>434</b>, 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> <em style='font-style: italic'>J. Exp. Med</em>. <b>190</b>, 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> <em style='font-style: italic'>Adv. Immunol</em>. <b>59</b>, 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> <em style='font-style: italic'>J. Immunol</em>. <b>169</b>, 1340-1348 (2002).</li></ol>

<p class="jove_content">In this procedure, it is shown how to initiate, grow, infect  Insect  cells and how to titer the baculovirus using these cells. The most important aspects of this procedure are maintenance of cells and to know the exact titer of baculovirus. Once you have  generated CD1 tetramers, they can be used as a powerful tool for analysis of glycolipid  reactive T cells.  Baculovirus systems are also used widely to produce recombinant proteins, including MHC class II tetramers so this  procedure has  a much wider applicability.</p>

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<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.

<p class="jove_content">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. </p><p class="jove_content">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.</p>

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.

<p class="jove_content">A Method to prepare Insect cells and infect them with baculovirus for the the purpose of production of recombinant mCD1d  proteinand generating mCD1d tetramers.</p>

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

Research

JoVE Journal

Biology

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 ...

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

En protokoll for Fag Display og Affinitetsutvelgelse Bruke rekombinante protein agn

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 <em>Agrobacterium</em>-mediated Transient Protein Production in <em>Nicotiana</em>

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

Optimalisering og utnyttelse av<em&gt; Agrobacterium</em&gt;-Mediert Transient Protein Produksjonen i<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

Effektiv produksjon og rensing av rekombinant mus Kindlin-3 fra insektceller for biofysiske studier

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

Gransker Protein-protein interaksjoner i levende celler ved hjelp Bioluminesens Resonance Energy Transfer

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

Rekombinant protein uttrykk for strukturbiologi i HEK 293F Suspension Cells: A Novel og tilgjengelig Approach

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 <em>Escherichia coli</em>

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

Grønn Fluorescent Protein-baserte Expression Screening av membran proteiner i<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

En komparativ analyse av rekombinant protein uttrykk i ulike Biofactories: Bakterier, insektsceller og plantesystemer

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

Nyreskade Mikroinjeksjon i Sebrafisk til Model Akutt nyreskade

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

Et grafisk brukergrensesnitt for programvareassistert sporing av proteinkonsentrasjon i dynamiske cellulære protrusjoner

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