摘要
目的优化BL21/pET-11 c/hIL-2-mGM-CSF的培养条件以提高目的蛋白的表达量。方法采用拉丁方试验设计方法进行培养基、诱导温度和IPTG使用浓度的综合比较试验,对电泳结果进行扫描后作统计分析,并对该试验所推导出的培养条件进行验证。以最佳的培养条件进行连续3次扩大培养,将其与常规培养条件进行比较。结果采用高浓度肉汤培养得到的蛋白相对表达量优于2×YT和LB培养基。42℃诱导与37℃诱导相比能显著提高蛋白表达量,而低至0.3 mmol/L的 IPTG可得到优于1.0mmol/L所能诱导得到的蛋白表达量。统计学分析表明优化的培养条件与常规培养条件相比能显著提高目的蛋白的相对表达量。扩大培养时,采用最佳的培养条件培养,蛋白的相对表达量比优化之前提高了5倍,表达量达到菌体总蛋白的29%以上。结论通过改良方法培养工程菌BL21/pET-11 c/hIL-2-mGM-CSF,hIL-2-mGM-CSF 蛋白以较高的水平表达,为下一步的蛋白纯化和功能分析奠定了基础。
Objective To optimize the culture condition of engineered E.coli to improve its expression efficiency of hIL-2-mGM-CSF protein, Methods 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 itsexpression 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, Results The cultures with TH broth yielded higher relative expression quantity of the target protein than those with 2xYT and LB medium. Compared with the induction temperature at 37 ℃, induction at 42 ℃ 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. Conclusion 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.
出处
《南方医科大学学报》
CAS
CSCD
北大核心
2006年第4期418-420,424,共4页
Journal of Southern Medical University
基金
国家重点基础研究发展规划(973)(2001CB510008)~~