RESUMO
Cystatins are reversible cysteine protease inhibitor proteins. They are known to play important roles in controlling cathepsins, neurodegenerative disease, and in immune system regulation. Production of recombinant cystatin proteins is important for biochemical and function characterization. In this study, we cloned and expressed human stefin A, stefin B and cystatin C in Escherichia coli. Human stefin A, stefin B and cystatin C were purified from soluble fraction. For cystatin C, we used various chaperone plasmids to make cystatin C soluble, as it is reported to localize in inclusion bodies. Trigger factor, GroES-GroEL, DnaK-DnaJ-GrpE chaperones lead to the presence of cystatin C in the soluble fraction. Immobilized metal affinity chromatography, glutathione sepharose and anion exchange chromatography techniques were employed for efficient purification of these proteins. Their biological activities were tested by inhibition assays against cathepsin L and H3 protease.
Assuntos
Cistatina A/genética , Cistatina A/isolamento & purificação , Cistatina B/genética , Cistatina B/isolamento & purificação , Cistatina C/genética , Cistatina C/isolamento & purificação , Catepsina L/antagonistas & inibidores , Catepsina L/química , Cistatina A/química , Cistatina A/metabolismo , Cistatina B/química , Cistatina B/metabolismo , Cistatina C/química , Cistatina C/metabolismo , Endopeptidases/química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/isolamento & purificação , Inibidores Enzimáticos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Humanos , Cinética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
Organisms living in water are inevitably exposed to periods of hypoxia. Environmental hypoxia has been an important stressor having manifold effects on aquatic life. Many fish species have evolved behavioral, physiological, biochemical and molecular adaptations that enable them to cope with hypoxia. However, the molecular mechanisms of hypoxia tolerance in fish, remain unknown. In this study, we used suppression subtractive hybridization to examine the differential gene expression in CAB cells (Carassius auratus blastulae embryonic cells) exposed to hypoxia for 24 h. We isolated 2100 clones and identified 211 differentially expressed genes (e-value