从毕赤酵母中提取外源重组蛋白的超声破碎条件及优化

Optimization of Ultrasonic Treatment for Recovery of Recombinant Protein from Pichia pastoris

建立适用于从毕赤酵母菌中提取外源重组蛋白的超声破碎方法。采用Western Blotting免疫印记技术检测从毕赤酵母菌中提取的外源人脂联素蛋白活性,并以外源人脂联素蛋白活性作为考察指标,采用单因素和L9(33)正交试验设计,对超声破碎条件进行优化。结果表明:在外源人脂联素蛋白活性最高的情况下,超声破碎条件为超声破碎功率450W、时间25min、时间间隔(工作时间:间歇时间)10:10(s/s),此时细胞破碎率为(67.8±2.1)%。若在超声破碎时添加1mmol/L PMSF,虽对细胞破碎率并无显著影响,但外源人脂联素蛋白活性将会明显增高。

In order to establish an appropriate ultrasonic treatment method for the recovery of recombinant protein from Pichia pastoris, the activity of human-adiponectin recombinant protein was evaluated by Western Blotting analysis. A L9（3^3） orthogonal array design based on single-factor tests was employed to optimize the conditions for ultrasonic treatment. The results showed that the optimal ultrasonic treatment conditions were ultrasonic power of 450 W, ultrasonic treatment time of 25 min and ultrasonic treatment interval of 10 s. Under the optimal ultrasonic treatment conditions, the breaking rate of cell walls was （67.8± 2.1）%. The activity of human-adiponectin recombinant protein could be significantly improved by adding 1 mmol/L of PMSF although the addition of PMSF had no effect on breaking rate of cell walls.