RESUMEN
OBJECTIVE: To investigate the limited digestion of recombinant staphylococcal enterotoxin C2 (SEC2-His)in different conditions. METHODS: The purified recombinant SEC2-His was treated with different reagents and the cleavage of rSEC2 molecule was observed by SDS-PAGE. RESULT: The cleavage occurred in positions Cys93-Cys110 of the disulfide loop. Complete auto-cleavage of recombinant SEC2 was observed in solution at 37degrees within 24 hrs, and that was accelerated under alkaline conditions. The auto-cleavage of the recombinant protein was inhibited in the presence of beta-ME (2%), PMSF (5-10 mmol/L), imidazole (1 mol/L) or crude E.coli lysate. Non-specific degradation of recombinant SEC2 was promoted with the increasing of the concentration of H(2)O(2). CONCLUSION: The recombinant SEC2-His is broken down in special site of protein, which may be associated with the protein structure.
Asunto(s)
Enterotoxinas/química , Proteínas Recombinantes de Fusión/química , Secuencia de Aminoácidos , Enterotoxinas/genética , Datos de Secuencia Molecular , Conformación Proteica , Estabilidad Proteica , Proteínas Recombinantes de Fusión/genéticaRESUMEN
AIM: To clone the gene of staphylococcal enterotoxin C2 and express it in the form of a soluble fusion protein in E. coli. Then the activation of SEC2 on mice lymphocyte and its lethal effects on tumor cells were studied. METHODS: Staphylococcus aureus SEC2 gene was cloned into GST gene fusion vector pGEX-4T-1. The resultant plasmid pGEX-4T-SEC2 was used to transform E. coli BL21, where the GST-SEC2 fusion protein was expressed efficiently. The rSEC2 protein was purified with Glutathione Sepharose 4B affinity column and digested with thrombin. The in vitro culture system was utilized to observe the activation of the SEC2 on mice lymphocyte and the lethal effects on tumor cells of the activated mice lymphocyte. RESULTS: The proper gene of SEC2 was cloned and purified rSEC2 was obtained. The MTT results indicated that rSEC2 have strong ability to stimulate mice lymphocyte to proliferate with a dose-dependent manner. With the proliferation of mice splenic lymphocyte, rSEC2 has a strong lethal effect on tumor cells B16, K562 and K562-AD. CONCLUSION: In this study, the gene of SEC2 was cloned and the rSEC2 protein was obtained, which had strong lethal effect on tumor cells B16, K562 and K562-AD.
Asunto(s)
Enterotoxinas/metabolismo , Activación de Linfocitos/efectos de los fármacos , Linfocitos/inmunología , Animales , Línea Celular Tumoral/efectos de los fármacos , Proliferación Celular , Clonación Molecular , Enterotoxinas/genética , Enterotoxinas/farmacología , Escherichia coli/genética , Escherichia coli/metabolismo , Femenino , Vectores Genéticos , Glutatión Transferasa/genética , Linfocitos/citología , Masculino , Ratones , Ratones Endogámicos ICR , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/farmacología , Bazo/citología , TransfecciónRESUMEN
Staphylococcal enterotoxins (SEs) are powerful superantigens that stimulate non-specific T-cell proliferation produced by Staphylococcus aureus and draw considerable attention as ideal drugs for cancer therapy. The filtrate of S. aureus culture has been used as ampul named Staphylococcal enterotoxin C injection in clinic for 10 years in China and proved to be effective. The superantigen SEC claimed to be the only active component without certifiable evidences. For further investigations of the active components of this injection and establishment of foundations for the development of novel anti-cancer drugs, in this research we extracted total DNA from S. aureus (FRI 1230), cloned, expressed and purified recombinant proteins of Staphylococcal enterotoxin M and N (rSEM and rSEN). The MTT assay of the purified rSEM and rSEN demonstrated that their abilities of stimulating T cells and inhibiting the proliferation of K562-ADM cells and B16 cells were equivalent to that of purified SEC2 in vitro. These findings suggested that SEC was not the only active component of Staphylococcal enterotoxin C injection and the effective procedure of expression and purification may be useful for mass productions of these therapeutically important proteins.