Generation of peptide-MHC class I complexes through UV-mediated ligand exchange.
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.
Rodenko
Boris
B
Division of Cellular Biochemistry, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX, Amsterdam, The Netherlands.
Toebes
Mireille
M
Hadrup
Sine Reker
SR
van Esch
Wim J E
WJ
Molenaar
Annemieke M
AM
Schumacher
Ton N M
TN
Ovaa
Huib
H
D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005805:Genes, MHC Class I / Q000235:genetics*; D008024:Ligands; D015394:Molecular Structure; D046912:Multiprotein Complexes / Q000138:chemical synthesis* / Q000235:genetics / Q000378:metabolism; D010455:Peptides / Q000378:metabolism*; D017510:Protein Folding; D014466:Ultraviolet Rays
D008024:Ligands; D046912:Multiprotein Complexes; D010455:Peptides