Chemical Engineering Discipline, School of Engineering, Monash University Malaysia, Jalan Lagoon Selatan, 47500 Bandar Sunway, Selangor, Malaysia; Advanced Engineering Platform, School of Engineering, Monash University Malaysia, Jalan Lagoon Selatan, 47500 Bandar Sunway, Selangor, Malaysia.
Department of Chemical Engineering/Graduate School of Biochemical Engineering, Ming Chi University of Technology, 24301 New Taipei City, Taiwan.
Int J Biol Macromol. 2020 Dec 1;164:4455-4465. doi: 10.1016/j.ijbiomac.2020.09.051. Epub 2020 Sep 13.
A stirred fluidized bed (SFB) ion exchange chromatography was successfully applied in the direct recovery of recombinant enhanced green fluorescent protein (EGFP) from the unclarified Escherichia coli homogenate. Optimal conditions for both adsorption and elution processes were determined from the packed-bed adsorption systems conducted at a small scale using the clarified cell homogenate. The maximal adsorption capacity and dissociation constant for EGFP-adsorbent complex were found to be 6.3 mg/mL and 1.3 × 10 mg/mL, respectively. In an optimal elution of EGFP with 0.2 M of NaCl solution (pH 9) and at 200 cm/h, the recovery percent of the EGFP was approximately 93%. The performances of SFB chromatography for direct recovery of EGFP was also evaluated under different loading volumes (50-200 mL) of crude cell homogenate. The single-step purification of EGFP by SFB recorded in a high yield (95-98%) and a satisfactory purification factor (~3 folds) of EGFP from the cell homogenate at 200 rpm of rotating speed.
搅拌流化床（SFB）离子交换色谱法成功应用于从未澄清的大肠杆菌匀浆物中直接回收重组增强型绿色荧光蛋白（EGFP）。从小规模的填充床吸附系统中使用澄清的细胞匀浆物进行实验，确定了吸附和洗脱过程的最佳条件。发现 EGFP-吸附剂复合物的最大吸附容量和解离常数分别为 6.3mg/mL 和 1.3×10mg/mL。在最佳的洗脱条件下（用 0.2 M 的 NaCl 溶液洗脱，pH9，流速为 200cm/h），EGFP 的回收率约为 93%。在不同的上样体积（50-200mL）的粗细胞匀浆下，SFB 色谱法对直接回收 EGFP 的性能也进行了评估。在 200rpm 的转速下，SFB 可一步法从细胞匀浆中以高收率（95-98%）和令人满意的纯化倍数（~3 倍）纯化 EGFP。
