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.

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5 minutes

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 only show you how to handle these cells and infect them with virtus for the purpose of generating more CD 1D tremors. Let's get started.First of all, I would like to show you how to the TN five insect cells, the high five cell line was originated from the ovarian cells of the cabbage looper insect. They are used in this protocol because they are growing serum free, medium adaptable to suspension culture and produce high levels of recombinant protein at five ml of insect express medium in 25 centimeter square tissue culture F flas. No, that express five comes without glutamine.So we need to add 10 ml of a hundred x glutamine pen strip, or glutamine alone into one liter of media. Now I am going to take the frozen vial of high five insect cells, thaw this in 37 degrees Celsius water bath immediately add high five insect cells to the flask and incubate for 30 minutes in 27 degrees Celsius. Incubator then aspirate the medium to get rid of the medium with DMSO gently without disturbing the cells attached on the bottom of the flask.Now we need to add five ml of fresh medium into the flas after hawing, we now need to expand the cells prior to infection with vir. Monitor the cells every day when flask is nearly 70%confident. Bring the cells in suspension by banging the flask on both sides.Transfer the cells to 1 75 centimeter square flask by adding 25 ml of insect express medium and five ml of cell culture split conference flask. One is to four or one is to five as needed. Do not let cells overgrow count the number of passages that you have split the cells.Do not go over passage number 30 because it may cause aging of cells resulting in cell lysis. Cells need to be split every other day. If we split, one is to five.When you reach the desired volume at the concentration of 1 million cells per ML infect cells, I generally grow up to two liter, which usually amount to 71 75 centimeter scale flask and approximately 25 ml per flask or four one liter ER flask and approximately 500 ml per flask. Growing cells up in ER flask is much faster at a hundred ml of culture and 400 ml of express five SFM medium and grow them in shaker at 1 25 RPM at 27 degrees Celsius temperature until we reach 1 million per ML cells of desired volume before infecting the cells, I would like to show you how to ti the belo virus and we will do it next. In order to tighter the virus, play 3000 to 5, 000 cells per well.Well in a flat bottom 96 well plate in 200 microliter of in insect express five SFM medium. Let the cells settle at 27 degrees Celsius for at least 30 minutes. Make the serial dilution of avius stock.One should do one is to tend dilution in one line of 96 value. Bottom plate using 250 microliter per well. Dilutions are made in express five SFM medium.Using a multichannel pipet transfer a fixed amount, usually 20 microliter of different dilutions to the cells culture at 27 degrees Celsius Incubator for seven days to avoid the evaporation from the edge rows. Wrap the edges of plate with fil after seven days. Check the plate and determine which dilution of virus gives a 50%infection.Then use a formula to compute the titer, which is expressed in PFU per ml. The formula is formed in most belo virus manuals. After ting, the virus, a required amount of virus to infect the cells for protein production, the multiplicity of infection should be between five to 10.For example, if the titer of mouse CD 1D belo viruses a hundred million plaque forming units per ml and we have 20 million cells per flask, we need to add one ml to two ml of belo virus to to infect the cells. The cells should be infected for four days. On day four after infection, the cells should be detached, floating, swollen, and forming sausage like sheep.Now it is time to recover the sup natant. Take the medium from infected flask transfer to 250 ml blue cap Falcon spinning bottles. They can be autoclaved and reused.Fill the bottles evenly and sent to fuge the cells in so GSA rotor at 2000 RPM, 20 minutes at four degree Celsius. Collect the SUP natin and proceed for further purification. I have just shown you how to initiate, grow and infect the high five insect cells and titer the balo virus.Using these cells, the most important aspects of this procedure is how to maintain the cells and how to know the exact titer of the baus. Once you have generated CD 1D tremors, they can be used as a powerful tool for analysis of glycolipid reactive T cells. This procedure for handling and ting the balo virus can be used for many other recombinant proteins as well.So that's it. Thanks for watching and good luck with your experiments.

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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.

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