RESUMO
An engineering E.coli strain, BL21 (DE3)/pGEX-4T hPTH (1-34), was constructed by oligonucleotide annealing and PCR amplifying the target gene, then ligating it with pGEX-4T-3 vector and transferring into BL21 host. The yield of soluble fusion protein of GST-hPTH(1-34) expressed from BL21(DE3)/pGEX-4T hPTH(1-34) is about 10 g/L after high-density, high expression culture and purification by affinity chromatography. Following the simple digestion of enterokinase, about 0.6 g/L intact hPTH (1-34) was harvested. The product is checked by HPLC MS and N-terminus sequence analysis. The purified recombinant hPTH(1-34) stimulated adenylate cyclase in rabbit renal cortical cell membranes to exactly the same extent as synthetic human parathyroid hormone standards, indicating that the recombinant product has full biological activity.
Assuntos
Enteropeptidase/metabolismo , Hormônio Paratireóideo/metabolismo , Fragmentos de Peptídeos/metabolismo , Adenilil Ciclases/metabolismo , Animais , Cromatografia Líquida de Alta Pressão , Escherichia coli/genética , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Hormônio Paratireóideo/genética , Hormônio Paratireóideo/isolamento & purificação , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/isolamento & purificação , Coelhos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Engineering E. coli strain, BL21 (DE3)/pGEX-4T-human Thymosin alpha 1, was constructed by oligonucleotide annealing and PCR amplifying the target gene, then ligating it with pGEX-4T-3 vector and transferring into BL21 host. The yield of fusion protein of GST-Thymosin alpha 1 expressed from BL21 (DE3)/pGEX-4T-thymosin alpha 1 is about 35%-40% of total protein after fermentation. Following the simple cut of thrombin or CNBr, about 0.2 g/L thymosin alpha 1 can be harvested. The product is checked by MS and activity test, which indicates that the recombinant product has full biological activity of native thymosin alpha 1.
