Preparative-scale purification and characterization of MHC class II monomers.
The MHC class II molecule is a heterodimeric glycoprotein consisting of one alpha and one beta polypeptide chain of almost identical molecular size. Recently it has been shown by others, and confirmed in our laboratory, that isolated monomers of murine MHC II molecules are capable of binding antigenic peptides like the alpha/beta intact heterodimer. In addition, preliminary results from our laboratory indicate that isolated single chain-peptide complexes of murine MHC class II molecules are capable of stimulating cloned T cells in an antigen specific manner. These results prompted us to isolate relatively large quantities of individual alpha and beta subunits of MHC II molecules for further in vitro and in vivo studies. Isolation of alpha and beta monomers proved to be difficult using conventional chromatographic methods. In this report we describe micro-preparative and preparative continuous flow electrophoresis methods by which milligram quantities of MHC II subunits can be purified. An optimal condition for the dissociation of heterodimeric MHC II into alpha and beta monomers was identified, and separation of human HLA DR2 and murine IAs monomers was accomplished. Both methods offer the resolving power of gel electrophoresis with the convenience of continuous sample elution. Purified MHC II subunits obtained by these methods were tested for their ability to bind antigenic peptides. Results presented in this study indicate that monomeric subunits of both human HLA-DR2 and murine IAs are equally active in specific binding of antigenic peptides like the native heterodimer.
Passmore
D
D
Anergen Inc., Redwood City, CA 94063.
Kopa
D
D
Nag
B
B
D000595:Amino Acid Sequence; D000818:Animals; D015802:HLA-DR2 Antigen / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D000949:Histocompatibility Antigens Class II / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D004676:Myelin Basic Protein / Q000737:chemistry / Q000276:immunology; D010455:Peptides / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D011485:Protein Binding
D015802:HLA-DR2 Antigen; D000949:Histocompatibility Antigens Class II; D004676:Myelin Basic Protein; D010455:Peptides