RESUMO
The cytokine TRAIL induces apoptosis in tumor cells of various origin without affecting normal cells. Clinical trials of TRAIL-receptor (DR4 and DR5) agonists (recombinant TRAIL or death receptors antibodies) have largely failed because most human tumors were resistant to them. Currently, a second generation of agents targeted at TRAIL-R with increased efficiency has been developed. To this end, we have developed DR5-B, a variant of TRAIL selectively interacting with DR5. We have developed a new efficient method for production of TRAIL and DR5-B using expression of these proteins in Escherichia coli strain SHuffle B. The proteins were isolated from the cytoplasmic fraction of cells and purified to a high degree of homogeneity using metal-affinity and ion-exchange chromatography. The protein yield was 211 and 173 mg from one liter of cell culture for DR5-B and TRAIL, respectively, which significantly exceeded the results obtained by other methods. DR5-B killed tumor cells of different origin more efficiently and rapidly compared with TRAIL. The resulting preparations can be used for the study of TRAIL signaling pathways and in preclinical and clinical trials as antitumor agents.
Assuntos
Receptores do Ligante Indutor de Apoptose Relacionado a TNF/biossíntese , Ligante Indutor de Apoptose Relacionado a TNF/biossíntese , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Cromatografia de Afinidade/métodos , Cromatografia por Troca Iônica/métodos , Ensaios de Seleção de Medicamentos Antitumorais , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Humanos , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/genética , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/isolamento & purificação , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/genética , Ligante Indutor de Apoptose Relacionado a TNF/isolamento & purificação , Ligante Indutor de Apoptose Relacionado a TNF/farmacologiaRESUMO
Clinical laboratory analysis of penetrating wounds of the eyes in children in 1990-1995 showed a tendency to an increase in the incidence of wound infection, which increased as high as to make 16.6%. Opportunistic bacteria predominated in the etiological structure of wound infection agents. They caused grave suppurative complications in the presence of decreased antibacterial resistance of the organism.
