Wen Qian, Ma Li, Wang Xiao-ning
Institute of Molecular Immunology, School of Biotechnology, Southern Medical University, Guangzhou 510515, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2006 Apr;26(4):418-20, 424.
To optimize the culture condition of engineered E.coli to improve its expression efficiency of hIL-2-mGM-CSF protein.
According to an orthogonal Latin square experiment design, the effects of the culture medium, temperature and IPTG concentration at different levels on the efficiency of the engineered E. coli were evaluated for its expression of hIL-2-mGM-CSF protein. The results of SDS-PAGE were analyzed with software and the culture conditions derived from the experimental results were tested in independent cultures. The optimal culture condition was used in three large-scale cultures and the results were compared with that of routine cultures.
The cultures with TH broth yielded higher relative expression quantity of the target protein than those with 2 x YT and LB medium. Compared with the induction temperature at 37 degrees C, induction at 42 degrees C significantly improved the expression efficiency of the target protein. IPTG at the concentration as low as 0.3 mmol/L produced better effect than 1.0 mmol/L IPTG. Statistical analysis suggested that the optimized culture conditions could obviously improve the expression efficiency of the target protein. Large scale cultures with the optimized culture condition resulted in a 5-fold improvement of the relative expression quantity of the protein, which accounted for over 29% of total bacterial protein.
The optimized culture condition of the engineered E. coli can remarkably increase the expression efficiency of hIL-2-mGM-CSF, which may facilitate the subsequent purification and functional study of the protein.
优化工程化大肠杆菌的培养条件，以提高其hIL-2-mGM-CSF蛋白的表达效率。
根据正交拉丁方实验设计，评估不同水平的培养基、温度和IPTG浓度对工程化大肠杆菌表达hIL-2-mGM-CSF蛋白效率的影响。用软件分析SDS-PAGE结果，并在独立培养中测试从实验结果得出的培养条件。将最佳培养条件用于三次大规模培养，并将结果与常规培养结果进行比较。
与使用2×YT和LB培养基的培养相比，使用TH肉汤的培养产生的目标蛋白相对表达量更高。与37℃诱导温度相比，42℃诱导显著提高了目标蛋白的表达效率。低至0.3 mmol/L的IPTG比1.0 mmol/L的IPTG产生的效果更好。统计分析表明，优化后的培养条件可明显提高目标蛋白的表达效率。采用优化培养条件的大规模培养使蛋白的相对表达量提高了5倍，占细菌总蛋白的29%以上。
工程化大肠杆菌的优化培养条件可显著提高hIL-2-mGM-CSF的表达效率，这可能有助于该蛋白后续的纯化和功能研究。
