Bioengineered soluble HLA-B7. Genesis, characterization, and occurrence of dimerization.
A soluble, secreted form of HLA-B7 was engineered by replacing the exons encoding the transmembrane and cytoplasmic domains of the B7 gene with a CI. The modified gene, gsB7, transfected into J27.2 or C1R cell lines, produced a secreted protein, sB7, serologically recognized as B7. Size fractionation showed one species of sB7 at the approximately 55 kD expected for an sB7 alpha-chain-beta 2m heteroduplex, and another at approximately 120 kD which had the same constituent chains and was a dimer of the 55-kD species. Dimer formation appeared to be related to protein concentration but not to disulfide bridging. The sB7 heavy chain on SDS-PAGE showed a doublet at approximately 39 and approximately 42 kD; enzyme analysis indicated that the two bands differed only by a carboxyl terminal polypeptide. Analysis of gsB7 transfectants' mRNA by Northern blots and PCR revealed message fully spliced or with retained CI, accounting for the 39- and 42-kD bands, respectively, and apparently untranslated message with I3 retained. sB7 was not detectable on the surface of gsB7 transfectants by CTLs, nor did it inhibit those CTLs. Production of the sB7 protein provides a ready, consistent source of soluble class I antigen for further study, including test materials for tolerogenicity studies in animal models.
Hiraki
D D
DD
Department of Pathology, Stanford University School of Medicine, California.
See-Tho
K
K
Filvaroff
E
E
Krishnaswamy
S
S
de Bello
W
W
Taidi-Laskowski
B
B
Grumet
F C
FC
D000595:Amino Acid Sequence; D002460:Cell Line; D002478:Cells, Cultured; D003601:Cytotoxicity Tests, Immunologic; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D005805:Genes, MHC Class I / Q000235:genetics; D005818:Genetic Engineering; D015793:HLA-B7 Antigen / Q000032:analysis / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011108:Polymers; D012333:RNA, Messenger / Q000032:analysis; D011994:Recombinant Proteins / Q000032:analysis / Q000302:isolation & purification; D012995:Solubility; D014162:Transfection
D015793:HLA-B7 Antigen; D011108:Polymers; D012333:RNA, Messenger; D011994:Recombinant Proteins