| Summary: | 碩士 === 明志科技大學 === 生化工程研究所 === 100 === Design of purification process for the His-enhanced green fluorescent protein (His-EGFP) from recombinant E. coli by immobilized metal affinity chromatography (IMAC) in a packed bed. A large amount of genetically engineered bacteria were cultured in a 5 L fermenter. Subsequently, the collected cells were suspended with buffer and disrupted by sonication method at 20 kHz and 4℃. The maximal release activity of His-EGFP from 25% (w/v) disrupted cells was found to be 8.39×105 (AU/mL). The commercial matrix of STREAMLINE Chelating was used to immobilize Ni2+ metal ion. The immobilized metal chelating adsorbent was employed to evaluate the dynamic binding capacity 5% breakthrough point for His-EGFP under four different parameters (i.e., pH, operating flow rate F, packed bed height H, and disrupted cell concentration Co) at one-factor-at-a-time experiments. Two-level factorial design was employed to confirm the important parameters. Moreover, the method of steepest ascent and central composite design were applied to find the optimal adsorption conditions. The optimal adsorption condition was found to be pH of 8.08, flow rate of 6.15 mL/min, disrupted cell concentration of 64.7% (ww/v), and packed bed height of 15.37 cm. The optimal conditions for the elution of adsorbed His-EGFP were further investigated in packed bed experiments and the optimal elution conditions were found to be 3 column volumes of 300 mM imidarzol containing 500 mM NaCl at flow rate of 300 cm/h. Finally, the optimal adsorption and elution conditions were employed for the purification of His-EGFP in packed beds. The results showed that the adsorption efficiency for His-EGFP was up to 86.68% and 97.94% at one column volume (30 ml feedstock) and 1/3 column volume (10 ml feedstock). The activity of His-EGFP were recovered with a yield of 85.45% (purification factor 3.09) and 97.21% (purification factor 3.20), respectively.
|
|---|
