ABSTRACT
Statistical experimental methods were used to optimize the medium for mass production of a novel laccase3 (Lac3) by recombinant Saccharomyces cerevisiae TYEGLAC3-1. The basic medium was composed of glucose, casamino acids, yeast nitrogen base without amino acids (YNB w/o AA), tryptophan, and adenine. A one-factor-at-a-time approach followed by the fractional factorial design identified galactose, glutamic acid, and ammonium sulfate, as significant carbon, nitrogen, and mineral sources, respectively. The steepest ascent method and response surface methodology (RSM) determined that the optimal medium was (g/L): galactose, 19.16; glutamic acid, 5.0; and YNB w/o AA, 10.46. In this medium, the Lac3 activity (277.04 mU/mL) was 13.5 times higher than that of the basic medium (20.50 mU/mL). The effect of temperature, pH, agitation (rpm), and aeration (vvm) was further examined in a batch fermenter. The best Lac3 activity was 1176.04 mU/mL at 25 °C, pH 3.5, 100 rpm, and 1 vvm in batch culture.
ABSTRACT
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.