Department of Developmental Immunology, La Jolla Institute for Allergy and Immunology
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Khurana, A., Kronenberg, M. A Method For Production of Recombinant mCD1d Protein in Insect Cells.. J. Vis. Exp. (10), e556, doi:10.3791/556 (2007).
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.
I. Thaw the Cells
Take the frozen vial of TN5 cells, and thaw it in a 37°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°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 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
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.
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°C. Collect the supernatant and proceed for further purification.
VI. Purification Of Recombinant mCD1d
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°C.
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.
|Express Five SFM||Serum Free Medium||Invitrogen||10486025|
|High Five TM||Insect Cells||Invitrogen||B855-02|
|Glutamine Pen strep||Antibiotics||Invitrogen||10378-016|
|25cm2 TC Flask||Plastic||Fisher Scientific||10-126-28|
|175 cm2 TC Flask||Plug seal flask||Fisher Scientific||10-126-8|
|Erlenmeyer Flask||Cell Culture Flask||Fisher Scientific||07 200 672|
|YM 30 Concenterator||30,000 MWCO||EMD Millipore||UFC 903096|
|FPLC equipment||GE Healthcare|
|BrA Enzyme and Buffer||Biotinylation Kit||Avidity||BIRA500|
|Ni-NTA Agarose Column||His Tag Protein Purification Column||GE Healthcare|
|Desalting column||To get Rod of Salt from Protein||GE Healthcare|
|MonoQ Column||Anion Exchange Chromatgraphy||GE Healthcare|
|S200 Column||Size Exclusion Chromatography||GE Healthcare|
|PE- Streptavidin||For conjugating Protein||Molecular Probes, Life Technologies||S-866|
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2. Kinjo, Y., Wu, D., Kim, G., Xing, G. W., Poles, M. A., Ho, D. D., Tsuji, M., Kawahara, K., Wong, C. H., and Kronenberg, M. (2005) Recognition of bacterial glycosphingolipids by natural killer T cells. Nature 434, 520-525
3. Naidenko, O. V., Maher, J. K., Ernst, W. A., Sakai, T., Modlin, R. L., and Kronenberg, M. (1999) . Binding and antigen presentation of ceramide-containing glycolipids by soluble mouse and human CD1d molecules . J. Exp. Med. 190, 1069-1080.
4. Porcelli, S. A. (1995). The CD1 family: A third lineage of antigen- presenting molecules. Adv. Immunol. 59, 1-98
5. Sidobre, S., and Kronemberg, M. (2002) . CD1 tetramers: A powerful tool for the analysis of glycolipid-reactive T cells. J. Immunol. 169, 1340-1348.