C-terminal epitope tagging facilitates comparative ligand mapping from MHC class I positive cells.
Purification of specific class I molecules prior to peptide ligand characterization is complicated by the presence of multiple class I proteins in most cell lines. Immortalized B, T, and tumor cell lines typically express endogenous HLA-A, -B, and -C; and most individuals from which the cell lines are derived are heterozygous at these loci. Antibodies specific for a particular HLA molecule may be used for purification, but allele-specific antibodies can be biased by ligands occupying the peptide-binding groove. Through the use of C-terminal tagging, we have developed a method of soluble HLA production such that downstream purification does not skew the peptide analysis of the examined molecule. Comparison of peptides eluted from HLA class I molecules with and without C-terminal tags demonstrates that addition of a tag does not abrogate the peptide binding specificity of the original molecule. Both pooled Edman sequencing and mass spectrometric sequencing identified no substantial differences in peptides bound by untailed, 6-HIS-tailed, and FLAG-tailed class I molecules, demonstrating that the peptide specificity of a given molecule is not distorted by either tag. This production methodology bypasses problems with isolation of specific molecules and permits ligand mapping and epitope discovery in a variety of pathogen-infected and tumor cell lines.
Hickman
H D
HD
University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA.
Batson
C L
CL
Prilliman
K R
KR
Crawford
D L
DL
Jackson
K L
KL
Hildebrand
W H
WH
D020816:Amino Acid Motifs; D000818:Animals; D019149:Bioreactors; D002851:Chromatography, High Pressure Liquid; D018604:Epitope Mapping / Q000379:methods*; D005822:Genetic Vectors; D006680:HLA Antigens / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015395:Histocompatibility Antigens Class I / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D013058:Mass Spectrometry; D010446:Peptide Fragments / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism*; D020539:Sequence Analysis, Protein; D013601:T-Lymphocytes / Q000737:chemistry; D014162:Transfection
D006680:HLA Antigens; D015395:Histocompatibility Antigens Class I; D008024:Ligands; D010446:Peptide Fragments