PURIFICATION OF IMMUNOLOGICALLY ACTIVE RECOMBINANT 21.5 KDA ISOFORM OF HUMAN MYELIN BASIC-PROTEIN

We have designed and expressed in bacteria a recombinant fetal form of human myelin basic protein (21.5 kDa isoform; rhMBP21.5), a candidate autoantigen in multiple sclerosis. An exon 2 insertion, carboxy-termi nal histidine tag and preferred bacterial codons differentiate the MBP 21.5 gene from that encoding the adult, brain-derived form of human MB P (18.5 kDa isoform; hMBP18.5). MBPs were expressed at high levels in E. coli and extracted from whole cells by simultaneous acid solubiliza tion and mechanical disruption. A nearly two-fold increase in recombin ant protein was detected in strains harboring MBP genes with bacterial preferred codons compared to genes containing human codons. The recom binant molecules were purified in two steps, first by reversed-phase c hromatographic separation and then by metal affinity chromatography. D imeric forms of recombinant MBP21.5 were detected under physiological conditions, however, substitution of a serine for the single cysteine at amino acid residue 81 resulted in only monomer formation. All forms of recombinant MBPs induced proliferative responses of human T lympho cytes specific for epitopes in MBP18.5 kDa. In contrast, human T cell lines that recognize an exon 2-encoded epitope of MBP responded to the 21.5 kDa isoform of MBP, but not the 18.5 kDa isoform.