Name: A Method For Production of Recombinant mCD1d Protein in Insect Cells. (Video) | JoVE Journal
Uploaded: 2007-12-10T22:00:00.000+00:00
Duration: 9 min 41 s
Description: Learn to produce recombinant mCD1d protein in High Five insect cells using baculovirus infection. This method generates mCD1d tetramers essential for NKT cell recognition in immunological research.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

playerTrialDesktop

I. Thaw the Cells
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 25cm2 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 5ml of Fresh medium.
II. Growing and Expanding the Cells
Monitor the cells every day. When flask is nearly 70% confluent, bring the cells in suspension by banging the flask on both sides and transfer the cells to 175cm2 flask by adding 21 ml of Insect express medium and 5ml of cell culture. Each T175 flask should have around 25-30ml medium as a&nbsp;final volume. Split confluent flask (approx 1 million/ml conc.) 1/4 or 1/5 as needed. Do not let cells overgrow. Count the number of passages that you have split the cells. Do not grow over passage number 30 because it may cause aging of cells resulting cell lysis. When you reach the desired volume at the concentration of 1 million/ml, infect the cells.
I generally grow up to 2 to 2.5 liters, which usually amounts to seventy 175cm2 flasks and approx. 25 to 30ml / flask, or five 1 liter Erlenmeyer flasks and approx. 400ml to 500 ml in each flask
Note: You either can grow cells in T-175 plug seal flask or Corning Erlenmeyer flask. Growing cells in Erlenmeyer Flasks is faster and easier.
III. Titering the Virus by End Point Dilution Method
<ol>
<li>Plate 3000-5000 cells per well in flat bottom 96 well plate in 200ul of Express five SFM medium. Let the cells settle at 27&deg;C for at least 30 minutes.</li>
<li>Make serial dilution of baculovirus stock. One should do 1/10 dilution in one line of 96 well U bottom plate, using 250ul per well. Dilutions are made in Express Five SFM medium. </li>
<li>Using a multichannel pipette, transfer a fixed amount (usually 20 ul) of different dilutions to the cells.</li>
<li>Culture at 27&deg;C for 7 days. To avoid the evaporation from the edge rows, wrap the edges of plate with parafilm.</li>
<li>After 7 days, check the plate and determine which dilution of virus gives a 50% infection - half the wells at this virus concentration are infected). Then use a formula to compute the titer, which is expressed in pfu/ml (pfu-plaque forming units). The formula is found in most baculovirus manuals.</li>
</ol>
IV. Infecting the cells
It is very important to know the Exact titer of virus. Add required amount of virus. For viral stock preparation the cells should be infected at Multiplicity of Infection (MOI) of not more than 1 (1pfu to 1High Five cell) Higher MOI leads to production of viral mutations. For protein production, usual amount is MO1 5-10.
Example:
The titer of mCD1d baculovirus = 1X108 pfu/ml
Add MOI=1 (to amplify the virus)= 20X10^6 cells/ flask / 1X10^8 pfu / ml=200 ul / flask
MOI= 5 to 10 (for protein production)= 1ml to 2ml For 20x10^6 cells/flask
On Day 4 after infection, look at the infected cells. They should be detached, floating, swollen and forming sausages.
V. Recovering Supernatants
Take the medium from infected flasks, and transfer to 250ml blue cap Falcon Spinning bottles. They can be autoclaved and reused. Fill the bottles evenly and centrifuge the cells in Sorvall GSA rotor at 200rpm, 20 minutes, 4&deg;C. Collect the supernatant and proceed for further purification.
VI. Purification Of Recombinant mCD1d
<ol>
<li>Dialysis and Concentration in 150mM Sodium Phosphate buffer (pH 7.4) </li>
<li>By Ni- NTA Agarose Chromatography, elute the protein mCD1d+b2m with 150mM Sodium phosphate +200mM Immidazole</li>
<li>Run Desalting column using 20mM Tris Hcl pH8 buffer to get rid of Immidazole</li>
<li>Run Anion Exchange Chromatography using MonoQ column to eliminate the remaining contaminants.</li>
<li>Concentrate to 1mg/ml using YM 30 concentrator (Millipore) </li>
<li>BirA enzymatic biotynlation of mCD1d according to manufacturer's protocol (Avidity) </li>
<li>The free biotin is eliminated by S200 column (size exclusion Chromatograpy) using phosphate buffered saline (PBS), pH7.4. An average of 2 to 3 mgs of biotynlated protein could be obtained from 2 liter of supernatant </li>
<li>Production of a-Galcer CD1d tetramer. </li>
</ol>
In order to load CD1d molecule with a-Galcer, soluble biotynlated CD1d-b2m is incubated overnight at room temperature with three fold molar excess of a-Galcer, dissolved in 0.5% Tween -20 in PBS, O.9% Sodium Chloride. Tetramers are generated by mixing of a-Galcer loaded CD1d monomers with 4 fold molar excess of PE - Conjugated streptavidin and incubating for 4 hours at room temperature. Store at 48&deg;C.

<ol><li>Benlagha, K., Weiss, A., Beavis, A., Teyton, L., Bendelac, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+identification+of+glycolipipd+antigen-specific+T+cells+using+fluorecent+CD1d+tetramers%5BTitle%5D)+AND+%22J.+Exp.+Med%22%5BJournal%5D)">In vivo identification of glycolipipd antigen-specific T cells using fluorecent CD1d tetramers.</a> J. Exp. Med. 191, 1895-1903 (2000).</li>
<li>Kinjo, Y., Wu, D., Kim, G., Xing, G. W., Poles, M. A., Ho, D. D., Tsuji, M., Kawahara, K., Wong, C. H., Kronenberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Recognition+of+bacterial+glycosphingolipids+by+natural+killer+T+cells%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Recognition of bacterial glycosphingolipids by natural killer T cells.</a> Nature. 434, 520-525 (2005).</li>
<li><a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Binding+and+antigen+presentation+of+ceramide-containing+glycolipids+by+soluble+mouse+and+human+CD1d+molecules%5BTitle%5D)+AND+%22J.+Exp.+Med%22%5BJournal%5D)">Binding and antigen presentation of ceramide-containing glycolipids by soluble mouse and human CD1d molecules.</a> J. Exp. Med. 190, 1069-1080 (1999).</li>
<li>Porcelli, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+CD1+family%3A+A+third+lineage+of+antigen-+presenting+molecules%5BTitle%5D)+AND+%22Adv.+Immunol%22%5BJournal%5D)">The CD1 family: A third lineage of antigen- presenting molecules.</a> Adv. Immunol. 59, 1-98 (1995).</li>
<li>Sidobre, S., Kronemberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CD1+tetramers%3A+A+powerful+tool+for+the+analysis+of+glycolipid-reactive+T+cells%5BTitle%5D)+AND+%22J.+Immunol%22%5BJournal%5D)">CD1 tetramers: A powerful tool for the analysis of glycolipid-reactive T cells.</a> J. Immunol. 169, 1340-1348 (2002).</li>
</ol>

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.

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

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

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.

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

Hi, I'm Ashra Corona from the lab of Dr.Mitch Kronenberg in the Department of Developmental Immunology at La Jolla Institute for Allergy and Immunology. Today I'm going to show you how to prepare high five insect cells and infect them with VIR for the purpose of generating MA CD 1D te tremors. CD 1D teters are used by our lab and by other for the recognition of NKT cells.VIR system are used widely to produce recombinant proteins including MS C class two TE tremors. So the procedure you will be shown has much wider applicability. This procedure involves initiating and growing up high five insect cells infect the cells with reovirus carrying CD 1D CDNA harvest supinate by centrifugation after four days of infection, lysis at concentration of protein in 150 millimeter sodium phosphate buffer, pH 7.4 purification of mouse CD 1D protein by nickel and T agros and iron exchange romme method concentration M CD one G protein to one me per ML using YM 30 concentrator where a enzymatic violation of mouse CD 1D by AVID D protocol, elimination of free BioTeam by S 200 column.And finally, production of alpha gel CD 1D Tet. Though the whole procedure involves all these steps, but today we will on

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

Research

JoVE Journal

Biology

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.

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

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

The MultiBac Protein Complex Production Platform at the EMBL

Kindlins are fundamental to cell adhesion through integrins but studies of them have been hampered by the difficulty encountered in expressing them recombinantly in bacterial hosts. We describe here methods for their efficient production in baculovirus-infected insect cells.

Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies

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

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

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

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

Nonlytic insect cell expression systems are underutilized for production, cellular trafficking/localization, and recombinant protein functional analysis. Here, we describe methods to generate expression vectors and subsequent transient protein expression in commercially available lepidopteran cell lines. The co-localization of Bemisia tabaci aquaporins with subcellular fluorescent marker proteins is also presented.

Transient Expression and Cellular Localization of Recombinant Proteins in Cultured Insect Cells

We report using an artificial intronic small RNA expression strategy to develop a non-defective recombinant Aedes aegypti densovirus (AaeDV) in vivo delivery system. A detailed procedure for the construction, packaging, and quantitative analysis of the rAaeDV vectors as well as for larval infection is described.

Use of a Recombinant Mosquito Densovirus As a Gene Delivery Vector for the Functional Analysis of Genes in Mosquito Larvae

This protocol describes a heat shock-induced protein expression system (pDHsp/V5-His/sf9 cell system), which can be used for either expressing foreign proteins or evaluating the anti-apoptotic activity of potential foreign proteins and their truncated amino acids in insect cells.

Transient Expression of Foreign Genes in Insect Cells (sf9) for Protein Functional Assay

Here, we present a protocol to produce house fly carboxylesterase proteins in vitro with a baculovirus mediated insect cell expression system and later functionally characterize their roles in metabolizing permethrin, thereby, conferring pyrethroid resistance by conducting cell-based MTT assay and in vitro metabolic studies.

Functional Characterization of Carboxylesterases in Insecticide Resistant House Flies, Musca Domestica

Here, we present the protocols for utilizing the insect cell and baculovirus protein expression system to produce large quantities of plant secreted proteins for protein crystallization. A baculovirus expression vector has been modified with either GP67 or insect hemolin signal peptide for plant protein secretion expression in insect cells.

Modifying Baculovirus Expression Vectors to Produce Secreted Plant Proteins in Insect Cells

The baculovirus expression vector system (BEVS) is a robust platform for expression screening and production of protein arginine methyltransferases (PRMTs) to be used for biochemical, biophysical, and structural studies. Milligram quantities of material can be produced for the majority of PRMTs and other proteins of interest requiring a eukaryotic expression platform.

Production of Recombinant PRMT Proteins using the Baculovirus Expression Vector System

Learn to produce recombinant mCD1d protein in High Five insect cells using baculovirus infection. This method generates mCD1d tetramers essential for NKT cell recognition in immunological research.

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

Explore More Videos