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Generation of peptide–MHC class I complexes through UV-mediated ligand exchange

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Major histocompatibility complex (MHC) class I molecules present peptide ligands on the cell surface for recognition by appropriate cytotoxic T cells. MHC-bound peptides are critical for the stability of the MHC complex, and standard strategies for the production of recombinant MHC complexes are based on in vitro refolding reactions with specific peptides. This strategy is not amenable to high-throughput production of vast collections of MHC molecules. We have developed conditional MHC ligands that form stable complexes with MHC molecules but can be cleaved upon UV irradiation. The resulting empty, peptide-receptive MHC molecules can be charged with epitopes of choice under native conditions. Here we describe in-depth procedures for the high-throughput production of peptide-MHC (pMHC) complexes by MHC exchange, the analysis of peptide exchange efficiency by ELISA and the parallel production of MHC tetramers for T-cell detection. The production of the conditional pMHC complex by an in vitro refolding reaction can be achieved within 2 weeks, and the actual high-throughput MHC peptide exchange and subsequent MHC tetramer formation require less than a day.

*Note: In the version of this article originally published online, the Reagent Setup listing for wash buffer should have read: “20 mM Tris pH 8, 100 mM NaCl.” This error has been corrected in the HTML and PDF versions of the article.

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Figure 1: UV-mediated peptide exchange.
Figure 2: Conditional peptide ligand.
Figure 3: Synthesis of the photolabile amino acid residue.
Figure 4: HLA A2.1–selective, UV-sensitive conditional MHC ligand containing a (2-nitro)phenylglycine residue.
Figure 5: Flow scheme of the various procedures involved in MHC peptide exchange.
Figure 6: Flow scheme of the assembly of biotinylated MHC class I monomers.
Figure 7: MHC class I tetramer formation.
Figure 8: ELISA setup and results.
Figure 9: Typical HPLC profile of an MHC PE-streptavidin titration.
Figure 10: Flow cytometry.

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  • 19 October 2006

    In the version of this article originally published online, the Reagent Setup listing for wash buffer should have read: “20 mM Tris pH 8, 100 mM NaCl.” This error has been corrected in the HTML and PDF versions of the article.


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Correspondence to Ton N M Schumacher or Huib Ovaa.

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Competing interests

The technology described in this manuscript is the subject of a patent application. Based on Netherlands Cancer Institute policy on management of intellectual property, M.T., H.O and T.N.M.S. would be entitled to a portion of received royalty income in case of future licensing.

Supplementary information

Supplementary Fig. 1

Synthetic scheme for the synthesis of N-Fluorenylmethyloxycarbonyl-(2-nitro)phenylglycine. (PDF 72 kb)

Supplementary Fig. 2

Standard titration curve. (PDF 54 kb)

Supplementary Methods (DOC 51 kb)

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Rodenko, B., Toebes, M., Hadrup, S. et al. Generation of peptide–MHC class I complexes through UV-mediated ligand exchange. Nat Protoc 1, 1120–1132 (2006).

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