RESUMEN
Large scale abstraction and isolation of bacterially synthesized, recombinant-DNA-derived, porcine growth hormone (r-pST) is described. The r-pGH is found in genetic engineering E. coli as the form of inclusion bodies. Pellet fraction which were mainly inclusion bodies, after cell breakage and centrifugation, were collected. Cell envelope components, such as protein, lipid, endotoxin and nucleic acids are selectively removed from the pellet fraction by an EDTA/lysozyme/deoxycholate extraction. Inclusion bodies were dissolved using 6 mol/L guanidine/HCl and air oxidation is then carried out in the presence of the guanidine/HCl. The Guanidine/HCl protein mixture were diluted by renaturation solution. Guanidine/HCl were removed by dialysis and then correctly refolded, oxidized r-pGH were obtained. Injection experiment of hypophysectomized rats proved r-pST with high native bioactivity was obtained.
Asunto(s)
Hormona del Crecimiento/aislamiento & purificación , Renaturación de Proteína , Animales , Bioensayo/métodos , Escherichia coli/genética , Hormona del Crecimiento/biosíntesis , Hormona del Crecimiento/química , Hormona del Crecimiento/genética , Hipofisectomía/métodos , Cuerpos de Inclusión/genética , Cuerpos de Inclusión/metabolismo , Pliegue de Proteína , Ratas , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , PorcinosRESUMEN
Human T-cell leukemia virus type-I (HTLV-I) has a post-transcriptional regulatory gene termed rex. We have designed the rex gene to express in E. coli. Synthesis of rex protein, p27rex, was examined by immunoblot analysis using anti-p27rex antibody. No difference in electrophoretic mobility in NaDadSO4-PAGE was observed between p27rex expressed in E. coli and in an HTLV-I-infected cell line, MT-2. Slower migration of p27rex, corresponding to a 27-kD protein, in NaDodSO4-PAGE when compared with the calculated molecular weight from the amino acid sequence (Mr = 20,367) is suggested to be caused not by post-translational modification, but by the intrinsic nature of the protein, which is rich in proline and arginine.