Optimization of culture conditions for the production of GST-EGFP fusion protein and study of its purification properties

• Main Authors: YANG,HSIAO-CHUN, 楊筱君
• Other Authors: Chang, Yu-Kaung
• Format: Others
• Language: zh-TW
• Published: 2009
• Online Access: http://ndltd.ncl.edu.tw/handle/12369026040133516414

碩士 === 明志科技大學 === 生化工程研究所 === 97 === In this study, early use of response surface methodology to find shake flask GST-EGFP optimized production conditions, shake flask culture, the best conditions for the start of pH 8.0, against 1% of the volume of bacteria, the initial temperature 37 ℃, speed of 230 rpm, when the OD600 reached 0.6-0.8 when induced by adding IPTG to final concentration of 1 mM, and reduce the culture temperature is 25 ℃, induction time for 20 hours. Shake flask to the best conditions for 6.6 L fermentation tank to zoom in culture, the cell is configured to collect 20% (w / v), broken by high pressure, the release of glutathione transferase (GST) activity up to to 82.45 (U / ml), green fluorescent protein (EGFP) for 858962.7 (AU / ml). Adsorption procedures in commercial use as an adsorption resin STREAMLINE Chelating carrier, selected the four metal ions (Cu 2 +, Zn2 +, Ni2 + and Co2 +) immobilized on the carrier to explore the different adsorption pH, types of immobilized metal ions and the density of metal ions on the target immobilized protein adsorption efficiency. Chelating resin was found in 20 mM pH 7 resin immobilized Ni2 + on the EGFP fluorescence intensity (4002107.34 AU / ml) and GST activity (85.36 U / ml) the maximum adsorption capacity. In order to obtain the best red EGFP and GST mentioned operation, desorption experiments, including (1) NaCl salts and competitive functional groups (imidazole) gradient of concentration on the desorption efficiency, and (2) to red on the desorption efficiency of flow impact, (3) filled with different amount of resin on the target protein, the effect of adsorption and desorption. The results showed that the resin in 40 ml filling volume, the target protein adsorption efficiency of 96%, stage desorption using 250 mM imidazole, the operation flow 150 cm / hr, EGFP recycling rate to 49.9%, and enhance the purity of about 1.23 times; Purification of GST is 73.0% recovery rate and purity of about 1.74 times to upgrade.