长链人胰岛素样生长因子-1在毕赤酵母中表达条件的优化

Optimization of condition for expression of long chain Arg3 human insulin-like growth factor-1 in Pichia pastoris

目的优化长链人胰岛素样生长因子-1(Long chain Arg3 human insulin-like growth factor-1,LR3IGF-1)在毕赤酵母中的表达条件。方法考察甲醇诱导浓度(0.25%、0.50%、0.75%、1.00%、1.25%和1.50%)、诱导温度(25、28、30℃)、pH值(4.75、5.00、5.25、5.50、5.75和6.00)及添加剂(0.1%的精氨酸、甘氨酸、组氨酸、吐温-20和吐温-80)对LR3IGF-1在毕赤酵母中表达的影响,优化LR3IGF-1的表达条件。以最佳表达条件在发酵罐中诱导表达LR3IGF-1,检测其表达量,并与初始发酵条件的表达量进行比较。结果甲醇浓度为0.75%及诱导温度为25℃时,LR3IGF-1的表达量最高,分别为76.5和98 mg/L;pH值为5.25,诱导24 h,LR3IGF-1的表达量较高,且降解程度低;添加吐温-20可使LR3IGF-1的表达量提高至271 mg/L。以优化的条件进行发酵,LR3IGF-1的表达量可达497 mg/L,较优化前(52 mg/L)提高了近9倍,且诱导时间从48 h缩短至24 h。结论优化了LR3IGF-1在毕赤酵母中的表达条件,为其规模化生产奠定了基础。

Objective To optimize the condition for expression of long chain Arg3 human insulin-like growth factor-1 （LR3IGF-1） in Pichia pastoris. Methods The effects of methanol concentration （0. 25%, 0. 50%, 0. 75%, 1. 00%, 1. 25% and 1. 50%）, temperature（25, 28 and 30~C）, pH value（4. 75, 5.00, 5. 25, 5.50, 5. 75 and 6.00） and additives （0. 1% Arg, Gly, His, Tween- 20 and Tween-80） on expression of LR3IGF-1 in 19. pastoris were investigated, based on which the condition for expression of LR3IGF- 1 was optimized. The expression of LR3IGF-1 in fermenter was induced under the optimized condition, and the expression level was determined and compared with that under original condition. Results The expression levels of LR3IGF-1 after induction with 0. 75% methanol and at 25℃ were 76. 5 and 98 mg/L respectively. The expression level of LR31GF-1 was high after induction at pH 5.25 for 24 h, with low degradation. After addition of Tween-20, the expression level increased to 271 mg/L. The expression level of LR3IGF-1 under optimized condition reached 497 mg/L, which increased by nearly 9 folds as compared with that before optimization of condition for expression, while the time for induction was shortened from 48 h to 24 h. Conclusion The condition for expression of LR3IGF-I in P. pastor/s was optimized, which laid a foundation of large-scale production of LR3IGF-1.