Efficient generation of major histocompatibility complex class I-peptide complexes using synthetic peptide libraries.
The use of synthetic random peptide libraries is a powerful technology for the study of many aspects of antigen presentation and peptide selection by major histocompatibility complex (MHC) molecules. Here we have used them in conjunction with a recombinant system to determine the peptide binding motifs of three classical class I MHC molecules of the laboratory rat: RT1-Aa, RT1-Au, and RT1-A1c. Described is a method for producing large amounts of soluble class I heavy and light chains in bacteria. Refolding RT1-Aa heavy chain (HC) with rat beta2-microglobulin (beta2m) in the presence of a specific peptide and the subsequent purification of the complex yielded conformationally correct material. This was assessed by gel chromatography, SDS-polyacrylamide gel electrophoresis, isoelectric focussing gel electrophoresis, enzyme-linked immunosorbent assay, and fluorescence-activated cell sorter analysis employing a previously unreported method utilizing a His-Tag affinity silica. By refolding RT1-Aa HC and rat beta2m around a random nonapeptide library and subjecting the resulting complex to acid elution of the bound peptides and pool sequencing, the peptide binding motif for this MHC class I molecule was determined. Results corresponded well with those previously determined from naturally bound peptides and in addition gave a clear and unambiguous signal for the C-terminal anchor residue. This method was then applied to determine the previously undescribed binding motifs for RT1-Au and RT1-A1c. For both molecules, the whole motif was confirmed from naturally bound peptides. We propose this method as an alternative way to obtain the whole class I MHC peptide motif, particularly when a specific antibody is unavailable and/or natural expression of the class I molecule of interest is low.
Stevens
J
J
Department of Immunology, The Babraham Institute, Cambridge CB2 4AT, United Kingdom. james.stevens@bbsrc.ac.uk
Wiesmüller
K H
KH
Barker
P J
PJ
Walden
P
P
Butcher
G W
GW
Joly
E
E
D000818:Animals; D001665:Binding Sites; D003198:Computer Simulation; D006649:Histocompatibility Antigens / Q000235:genetics / Q000378:metabolism*; D015395:Histocompatibility Antigens Class I / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D009842:Oligopeptides / Q000378:metabolism*; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D019151:Peptide Library; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D001613:beta 2-Microglobulin / Q000235:genetics / Q000378:metabolism
D006649:Histocompatibility Antigens; D015395:Histocompatibility Antigens Class I; D009842:Oligopeptides; D010446:Peptide Fragments; D019151:Peptide Library; D011993:Recombinant Fusion Proteins; D001613:beta 2-Microglobulin; C038353:histocompatibility antigens RT, rat