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#149
PRODUCTION AND PURIFICATION OF SOLUBLE CLASS I MOLECULES USING A VLDL-RECEPTOR TAG.
Wilfried Bardet , Heather D. Hickman , Angela D. Luis and William H. Hildebrand . Oklahoma city OK, USA, Oklahoma Universtiy Health Sciences Center, 73104, Microbiology Immunology .

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MHC class I molecules present intracellularly-derived peptides to immune effector cells. In order to discover peptide epitopes, individual class I molecules must be isolated. Our laboratory has developed a technique for large-scale purification of class I molecules that have been secreted from tumor and virus-infected cell lines utilizing soluble HLA and hollow-fiber bioreactor culture. However, purification of the desired class I molecule from cells expressing multiple HLA can be complicated by the cross-reactivity of antibodies with multiple HLA. We previously demonstrated that the carboxy-terminal tailing of class I molecules does not alter the binding of peptides to the molecule, and we next began to test antibody recognition of various tails to facilitate purification of the tagged class I. ATCC recently released a hybridoma raised against the peptide SVVSTDDDLA corresponding to the 10 carboxy-terminal amino acids of the rat VLDL-receptor (VLDLr). This amino acid sequence was added through PCR to the C-terminus of A*0201, creating a soluble molecule with a C-terminal VLDLr tag (A*0201-VLDLr). Cell line 721.221 was transfected by electroporation with A*0201-VLDLr, and the resulting transfectants yielded more than 100 mg of A*0201 in a single experiment. Antibody reactivity was confirmed by WESTERN BLOTTING and ELISA, and recombinant A*0201-VLDLr was subsequently recovered using anti-VLDLr affinity chromatography. The VLDLr tailing of HLA proteins therefore represents a robust system for production and purification of single class I molecules from a mixture. This technology will facilitate epitope discovery methodologies for vaccine, and drug discovery.