Direct recovery of enhanced green fluorescent protein from unclarified Escherichia coli homogenate by using stirred fluidzied bed adsorption technique

• Main Authors: Lu Chung-Hsuan, 陸崇軒
• Other Authors: Chang Yu-Kaung
• Format: Others
• Language: zh-TW
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/20973642810046493499

碩士 === 明志科技大學 === 生化工程研究所 === 95 === An improved procedure was developed involving a stirred fluidized bed (SFB) adsorption for direct recovery of recombinant enhanced green fluorescent protein (EGFP) from unclarified E. coli homogenates. The maximal binding capacity of STREAMLINE DEAE adsorbent for EGFP was observed at pH 9. A typical adsorption isotherm curve for EGFP in a crude E. coli homogenate was well correlated with the Langmuir model. The maximal binding capacity for EGFP and dissociation constant for EGFP-adsorbent complex were 6.3mg/ ml and 1.3x10-3 mg/ ml respectively. Optimal conditions for elution scheme were further determined in small packed bed experiments conducted with clarified E.coli homogenate under the various operating conditions. The recovery yield for the EGFP was approximately 93% by using 0.2 M NaCl (pH 9) and 10 fold adsorbent volumes at liquid velocity of 200 cm/h. The operating conditions were chosen for use in further purification process. Finally, the experiments were carried out to assess the feasibility of using SFB chromatography for direct recovery of EGFP from different loading volumes (50-200 ml) of highly crude E.coli homogenate (pH 9). It was found to be successful in achieving purification of EGFP in a high yield of 95-98% and the purification factor was approx. 3 in a single step under these loading volumes of E. coli homogenates at a rotating speed of 200 rpm.