RESUMEN
To express Pleurocidin in Escherichia coli and to enhance the secretory efficiency of the fusion protein, the gene encoding Pleurocidin was ligated with Cherry DNA sequence via blunt-end ligation. Then this fusion gene was cloned into pET22b (+) vector and the recombinant plasmid was transformed into E. coli BL21 (DE3). Lactose was used to induce expression of fusion protein. The recombinant plasmid pET22b (+) -CP was successfully constructed and high-level expression of fusion protein was induced with lactose. Statistics showed that addition of glycine after 16 h of induction significantly enhanced the secretory efficiency of the fusion protein. After hydrolysis of the fusion protein by diluted hydrochloric acid and some further purification steps, r-Pleurocidin was obtained with antibacterial activity against E. coli DH5α and Bacillus subtilis BS168. In conclusion, the fusion protein was expressed in E. coli and biologically active r-Pleurocidin was obtained after hydrochloric acid cleavage and purification.
Asunto(s)
Animales , Clonación Molecular , Escherichia coli , Metabolismo , Proteínas de Peces , Lenguado , Proteínas Recombinantes de FusiónRESUMEN
The G13 domain derived from granulysin shows high antimicrobial activities against Gram-positive and Gram-negative bacteria but does not lyse Jurkat cells or liposomes. To explore a new approach for high expression of the G13 domain, we fused the sequence encoding G13 to thioredoxin (Trx) gene to construct the recombinant expression vector (pThioHisA-G13). A cyanogen bromide (CNBr) cleavage site was introduced between the Trx and G13 to facilitate final release of the recombinant G13. The recombinant expression vector, pThioHisA-G13, was transformed into E. coli BL21 (DE3). Upon induction by IPTG Trx-G13 fusion protein was expressed and took the form of inclusion bodies counting 58% (W/W) of total cellular proteins. The inclusion body was solved by urea (8 mol/L) and then cleaved by CNBr. We purified the recombinant peptide G13 by one-step cation exchange chromatography. Results of agarose diffuse assay analysis indicated that the recombinant G13 exhibited antibacterial activity. The procedure described in this study will provide a reliable and simple method for highly efficient production of some cationic antimicrobial peptides.
Asunto(s)
Antiinfecciosos , Metabolismo , Antígenos de Diferenciación de Linfocitos T , Genética , Bromuro de Cianógeno , Farmacología , Escherichia coli , Genética , Metabolismo , Subunidades alfa de la Proteína de Unión al GTP G12-G13 , Genética , Cuerpos de Inclusión , Metabolismo , Estructura Terciaria de Proteína , Genética , Proteínas Recombinantes de Fusión , Genética , Tiorredoxinas , Genética , TransfecciónRESUMEN
Alzheimers disease is the most common cause of progressive decline of mental function. Recent years there is a large development in the early diagnosis and therapeutic progress in Alzheimer disease. The article reviews the progress in the pathogenesis, early diagnosis and new therapies in Alzheimers disease.