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

Summary

A Method to prepare Insect cells and infect them with baculovirus for the the purpose of production of recombinant mCD1d proteinand generating mCD1d tetramers.

Abstract

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.

Protocol

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 ....

Discussion

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.

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Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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
alpha-Galcer	Glycolipid
PE- Streptavidin	For conjugating Protein	Molecular Probes, Life Technologies	S-866