RESUMO
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.
Assuntos
Antígeno HLA-B7/genética , Sequência de Aminoácidos , Linhagem Celular , Células Cultivadas , Testes Imunológicos de Citotoxicidade , Eletroforese em Gel de Poliacrilamida , Expressão Gênica , Genes MHC Classe I/genética , Engenharia Genética , Antígeno HLA-B7/análise , Antígeno HLA-B7/isolamento & purificação , Humanos , Dados de Sequência Molecular , Peso Molecular , Polímeros , RNA Mensageiro/análise , Proteínas Recombinantes/análise , Proteínas Recombinantes/isolamento & purificação , Solubilidade , TransfecçãoRESUMO
Peptides and other bioactive materials can be purified from complex biological sources by reverse-phase high-performance liquid chromatography (RP-HPLC), provided the mixture is suitably prepared before injection onto an HPLC system. Ultrafiltration offers a convenient and rapid sample preparation technique with numerous advantages over alternative methods such as conventional gel filtration chromatography. We demonstrate the use of ultrafiltration as an HPLC sample preparation step in the purification of peptides bound to class I major histocompatibility complex (MHC-I) membrane proteins. When ultrafiltration was performed with a Centricon-10 ultrafiltration device, peptides were efficiently separated from the alpha (45 kDa) and beta 2m (12 kDa) chains of MHC-I proteins and could be subjected to HPLC without further treatment. Furthermore, even samples as crude as whole cell lysates or supernatants could be prepared for HPLC in a single ultrafiltration step, affording a remarkably straightforward route to the purification of biologically important peptides.
