RESUMEN
Transforming growth factor-j31 (TGF-p1) has potential for therapeutic use in common clinical conditions for which there are no adequate pharmacological agents. However, in vivo studies using TGF-p1 were hindered by high price of this cytokine. As a first step towards large scale purification of TGF-p1, it was purified in a small scale (10 unit platelets) from human platelets by four purification steps: platelet extraction, gel filtration, cation exchange chromatography, and reversed phase high performance liquid chromatography (HPLC). A single protein band with a molecular weight of 25 Kd corresponding to purchased TGF-p1 (R8D Systems) was confirmed by silver staining after SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of eluant from reversed phase HPLC. Recovery (%) of each step was about 50-60%, resulting in the final recovery of 20% based on the detection by a sandwich ELISA. Approximately, 3.7 p,g of purified TGF-p1 was obtained from 18 pg of platelet extracts. This result was confirmed by receptor (TGF-j31 type II) ELISA and bioassay using a mink lung epithelial'cell line (MV1LU). Further, in vitro characterization study showed that purified TGF-p1 inhibits G1/S transition of LPS-activated murine spleen B cells and increases surface IgA expression by the same cell population, which are typical activities of TGF-p1 in B cell differentiation. Taken together, the results from the present study reveals that purified TGF-p1 is fully biologically active and our purification methodology could be usbful to obtain a large scale of recombinant TGF-p1 in the future.
Asunto(s)
Humanos , Linfocitos B , Bioensayo , Plaquetas , Ciclo Celular , Diferenciación Celular , Cromatografía , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Cromatografía Liquida , Electroforesis , Ensayo de Inmunoadsorción Enzimática , Inmunoglobulina A , Pulmón , Visón , Peso Molecular , Tinción con Nitrato de Plata , Bazo , Factores de Crecimiento TransformadoresRESUMEN
No abstract available.