A chaperone-assisted high yield system for the production of HLA-DR4 tetramers in insect cells.
MHC tetramers have become essential tools for the analysis of antigen specific responses of CD8+ and CD4+ T cells. However, the use of MHC class II tetramers is hampered by the relatively low yields of most current expression systems. We have devised an insect cell/baculovirus expression system in which yields of 50-70 mg of recombinant HLA-DR4 molecules, with or without covalently linked peptide, per liter of insect cell supernatant, are routinely obtained. These yields are rendered possible by an optimized design and use of DRalpha and DRbeta expression cassettes and by co-expression of a housekeeping chaperone of the endoplasmic reticulum, calreticulin, which, due to its co-secretion, increases secretion of HLA-DR molecules two- to threefold. A tetramer produced in the system specifically was shown to stain an HLA-DR4 restricted T cell line obtained from a healthy donor by in vitro priming, but which recognizes a type I diabetes autoantigen. Co-expression of chaperones may represent a general strategy for enhancing yields of recombinant proteins expressed in insect cells and facilitate production of MHC class II tetramers in the future.
Institut National de la Santé et de la Recherche Médicale Unité 580, Hôpital Necker, 161 rue de Sèvres, 75015 Paris, France.
Jean-Marie
JM
Fourneau
Hélène
H
Cohen
Peter M
PM
van Endert
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae; D001483:Base Sequence; D037281:Calnexin / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology; D037282:Calreticulin / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology; D017931:DNA Primers; D005434:Flow Cytometry; D005822:Genetic Vectors; D015804:HLA-DR4 Antigen / Q000096:biosynthesis* / Q000737:chemistry / Q000187:drug effects; D006801:Humans; D007313:Insecta; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000138:chemical synthesis*; D013601:T-Lymphocytes / Q000276:immunology*
D037282:Calreticulin; D017931:DNA Primers; D015804:HLA-DR4 Antigen; D011994:Recombinant Proteins; D037281:Calnexin