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-terminal histidine tag and preferred bacterial codons differentiate the MBP21.5 gene from that encoding the adult, brain-derived form of human MBP (18.5 kDa isoform; hMBP18.5). MBPs were expressed at high levels in E. coli and extracted from whole cells by simultaneous acid solubilization and mechanical disruption. A nearly two-fold increase in recombinant protein was detected in strains harboring MBP genes with bacterial preferred codons compared to genes containing human codons. The recombinant molecules were purified in two steps, first by reversed-phase chromatographic separation and then by metal affinity chromatography. Dimeric 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 lymphocytes 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.
Alexion Pharmaceuticals, Inc., New Haven, CT 06511, USA.
S H
SH
Nye
C M
CM
Pelfrey
J J
JJ
Burkwit
R R
RR
Voskuhl
M J
MJ
Lenardo
J P
JP
Mueller
D000328:Adult; D000595:Amino Acid Sequence; D001483:Base Sequence; D005314:Embryonic and Fetal Development / Q000276:immunology; D005091:Exons / Q000276:immunology; D005822:Genetic Vectors; D006801:Humans; D007536:Isomerism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D004676:Myelin Basic Protein / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification*
D004676:Myelin Basic Protein; D011994:Recombinant Proteins