RESUMEN
Fibroblast growth factor 10 (FGF10) is a member of the FGF superfamily. It exhibits diverse biological functions, and is extensively used for fundamental research and clinical applications involving hair growth, tissue repair, and burn wounds. Oil bodies, obtained from oil seeds, have been exploited for a variety of biotechnology applications. The use of oil bodies reduces purification steps and costs associated with the production of heterogonous proteins. Here, recombinant human FGF10 (rhFGF10) was expressed in safflower (Carthamus tinctorius L.) seeds using oilbody-oleosin technology. A plant expression vector, pOTBar-oleosin-rhFGF10, was constructed and introduced into safflower using Agrobacterium tumefaciens transformation, and mature safflower plants were obtained by grafting. Oleosin-rhFGF10 was successfully transformed and expressed in safflower seeds and inherited to the T3 generation. Moreover, MTT assays demonstrated that oil bodies expressed oleosin-FGF10 had a dose-dependent effect on cellular proliferation. In conclusion, this may provide a method of producing oleosin-rhFGF10, and help us meet the increasing pharmacological demands for the protein.
Asunto(s)
Carthamus tinctorius/genética , Factor 10 de Crecimiento de Fibroblastos/genética , Vectores Genéticos/química , Proteínas de Plantas/genética , Semillas/genética , Agrobacterium tumefaciens/genética , Agrobacterium tumefaciens/metabolismo , Animales , Linfocitos B/citología , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Carthamus tinctorius/química , Carthamus tinctorius/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Factor 10 de Crecimiento de Fibroblastos/biosíntesis , Factor 10 de Crecimiento de Fibroblastos/aislamiento & purificación , Factor 10 de Crecimiento de Fibroblastos/farmacología , Expresión Génica , Vectores Genéticos/metabolismo , Humanos , Gotas Lipídicas/química , Gotas Lipídicas/metabolismo , Ratones , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/farmacología , Semillas/química , Semillas/metabolismoRESUMEN
A rapid and efficient expression and purification system has been developed for large-scale production of biologically active recombinant human keratinocyte growth factor-2 (rhKGF-2). The gene encoding human KGF-2 was cloned into the expression vector pET3c and transformed into Escherichia coli BL21(DE3)/pLys S. Under optimal conditions in a 30-l fermentor, the average bacterial yield and the average expression level of rhKGF-2 of three batches were up to 732 g and 32%, respectively. The recombinant protein was purified by cation exchange and heparin-affinity chromatography. One hundred and sixty five milligrams of pure rhKGF-2 was achieved per liter culture. A preliminary biochemical characterization of purified rhKGF-2 was performed by Western blotting and mitogenic activity analysis, and the results demonstrated that purified rhKGF-2 could react with anti-human KGF-2 antibody and stimulate the proliferation of HaCat cells.
Asunto(s)
Escherichia coli/genética , Factor 10 de Crecimiento de Fibroblastos/aislamiento & purificación , Factor 10 de Crecimiento de Fibroblastos/metabolismo , Expresión Génica , Proteínas Recombinantes/metabolismo , Biomasa , Línea Celular , Escherichia coli/metabolismo , Fermentación , Factor 10 de Crecimiento de Fibroblastos/genética , Vectores Genéticos/genética , Humanos , Mitógenos/genética , Mitógenos/aislamiento & purificación , Mitógenos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
Keratinocyte growth factor-2 (KGF-2) is a member of the fibroblast growth factor family. The full-length human KGF-2 coding sequence, gained by synthesizing, was cloned into the pPICZalphaA vector in frame with the yeast alpha-factor secretion signal under the transcriptional control of the AOX promoter and integrated into Pichia pastoris strain GS115. In shake-flask culture induced with methanol, the rhKGF-2 content was about 17.5% of the total secreted proteins. Under the optimal conditions, stable production of rhKGF-2 around 1.0g/l was achieved. The recombinant protein was purified by heparin affinity chromatography. A preliminary biochemical characterization of purified rhKGF-2 was performed both by Western blot analysis and biological activity analysis, and the result demonstrated that the recombinant KGF-2 was expressed successfully.
Asunto(s)
Factor 10 de Crecimiento de Fibroblastos/biosíntesis , Factor 10 de Crecimiento de Fibroblastos/genética , Pichia/genética , Pichia/metabolismo , Secuencia de Bases , Biotecnología , Western Blotting , Cartilla de ADN/genética , Factor 10 de Crecimiento de Fibroblastos/aislamiento & purificación , Expresión Génica , Vectores Genéticos , Humanos , Plásmidos/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
To clone KGF-2 gene, get hKGF-2 protein and detemine its activity. The cNDA of human KGF-2 was isolated from fetal lung by RT-PCR and cloned into pBV220 plasmid. The recombinant pBV220-hKGF-2 plasmid was transformed into E. coli (BL21), induced at 42 degrees C for the expression of hKGF-2. Recombinant human KGF-2 was purified from the ultrasonic-treated BL21 by heparin-Sepharose CL-6B treated column chromatography and cation exchange column chromatography. MTT method was used for the determination of its biological activity. SDS-PAGE showed that rhKGF-2 was expressed in E. coli BL21 as soluble protein of approximately 20kD. The rhKGF-2 protein can stimulate the proliferation of NIH3T3 cells significantly from 1 ng/mL to 10 ng/mL. HKGF-2 cDNA wasclned and highly expressed in E. coli BL21 and the purified rhKGF-2 showed the mitogenic activity on NIH3T3 cells.
