RESUMO
A hybrid gene consisting of the sequences coding for the signal peptide of human growth hormone and the mature form of interleukin-1 beta (IL-1 beta) was chemically synthesized. This sequence was inserted into a eukaryotic expression vector and introduced into Chinese hamster ovary cells. The resulting stably transformed cell lines produced large amounts of recombinant IL-1 beta, which was secreted into the culture medium mainly as a 22-kDa form. Expression in the presence of tunicamycin, an inhibitor of N-glycosylation, led to the complete disappearance of the 22-kDa form and the appearance of a new form of 17.5 kDa, indicating that the hybrid protein had been both processed and N-glycosylated. However, transformed cells producing mature IL-1 beta without a signal peptide produced the predicted 17.5-kDa nonglycosylated form. These results suggest that fusion to a heterologous leader sequence allowed IL-1 beta to be translocated across the membrane of the endoplasmic reticulum and to be transported and secreted by the exocytotic pathway.
Assuntos
Hormônio do Crescimento/genética , Interleucina-1/genética , Sinais Direcionadores de Proteínas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Clonagem Molecular , Cricetinae , Expressão Gênica , Glicosilação , Humanos , Interleucina-1/metabolismo , Dados de Sequência Molecular , Testes de Precipitina , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Tunicamicina/farmacologiaRESUMO
To assess the biological activity and pharmacokinetic properties of nonglycosylated ricin A-chain (RA), we have obtained the polypeptide following expression of a synthetic 842-bp RA gene in Escherichia coli. Expression of the gene was carried out using the phage T5 PN25 promoter fused to the E. coli lac operator. The RA polypeptide was synthesized in a completely soluble form and was purified in one step by immunoabsorption. It was shown to be as cytotoxic for a human cell line as both native RA and chemically deglycosylated native RA. Reconstituted whole ricin and an immunotoxin containing the recombinant RA were also biologically active. Immunotoxins made with recombinant and deglycosylated RA had similar clearance rates in vivo showing, after a short period of rapid elimination, stabilities far higher than that of an immunotoxin made with native RA. Our results show that the complete elimination of sugar side chains from the RA is not sufficient to entirely eradicate the rapid initial in vivo clearance of RA-based biologicals.
Assuntos
Escherichia coli/genética , Genes Sintéticos , Imunotoxinas/genética , Ricina/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular/efeitos dos fármacos , Glicosilação , Humanos , Imunotoxinas/farmacocinética , Óperon Lac , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Ricina/farmacocinéticaRESUMO
Plasma desorption (PD) mass spectra of normal deoxyribo-oligonucleotides and of neutral methylphosphonate deoxyribo-oligonucleosides are examined and discussed. Molecular ions of oligonucleotides up to nonamer have been observed for neutral species. It is also shown that PD mass spectra can be used to monitor chemical modifications of oligonucleotides, such as the covalent binding of an organic fluorescent probe, along the synthesis process.
