RESUMO
Recombinant human platelet-derived growth factor-BB (rhPDGF-BB) is used to treat full-thickness diabetic ulcers and is being investigated for use in other chronic ulcers, non-healing wounds, and periodontal defects. A simple, novel method for expression and purification of rhPDGF-BB from Escherichia coli is now described. This method produces the dimeric protein in high yield (10-12 mg/g wet cell mass) and with a purity >95%. rhPDGF-BB was exclusively found in inclusion bodies (IBs) representing approx. 30% of the total cell proteins. The IBs were extracted and the monomer purified by RP-HPLC. The purified rhPDGF-B monomer was then refolded using Tris buffer and subsequently dimerized to produce biologically active rhPDGF-BB. This product was composed of two polypeptide chains, each approx. 12 kDa. The final product exhibited specific activity in a fibroblast proliferation assay indistinguishable from that of the WHO reference standard.
Assuntos
Fibroblastos/efeitos dos fármacos , Engenharia de Proteínas/métodos , Becaplermina , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Clonagem Molecular/métodos , Escherichia coli/genética , Escherichia coli/metabolismo , Fibroblastos/citologia , Fibroblastos/fisiologia , Melhoramento Genético/métodos , Humanos , Fator de Crescimento Derivado de Plaquetas/administração & dosagem , Fator de Crescimento Derivado de Plaquetas/química , Fator de Crescimento Derivado de Plaquetas/genética , Fator de Crescimento Derivado de Plaquetas/metabolismo , Proteínas Proto-Oncogênicas c-sis , Proteínas Recombinantes/metabolismoRESUMO
Increasing therapeutic applications for recombinant human interferon-gamma (rhIFN-gamma), an antiviral proinflammatory cytokine, has broadened interest in optimizing methods for its production and purification. We describe a reversed phase chromatography (RPC) procedure using Source-30 matrix in the purification of rhIFN-gamma from Escherichia coli that results in a higher yield than previously reported. The purified rhIFN-gamma monomer from the RPC column is refolded in Tris buffer. Optimal refolding occurs at protein concentrations between 50 and 100 microg/ml. This method yields greater than 90% of the dimer form with a yield of 40 mg/g cell mass. Greater than 99% purity is achieved with further purification over a Superdex G-75 column to obtain specific activities of from 2 x 10(7) to 4 x 10(7)IU/mg protein as determined via cytopathic antiviral assay. The improved yield of rhIFN-gamma in a simple chromatographic purification procedure promises to enhance the development and therapeutic application of this biologically potent molecule.
