TAT-SOD在长期保藏的重组毕赤酵母中表达的条件优化

Optimization of TAT-SOD Expression Conditions in Long-Term Preserved Recombinant Pichia pastoris

摘要:

TAT-SOD是TAT蛋白转导结构域与SOD的融合蛋白质。作者以2007年构建的产TAT-SOD重组毕赤酵母菌为出发菌株，通过摇瓶实验研究不同温度、初始pH、诱导剂浓度等因素对蛋白质表达的影响。通过比色分析法测定菌体浓度、超氧阴离子自由基清除法测定酶活、SDS-PAGE分析目的蛋白质TAT-SOD的浓度；并采用定量PCR法分析表达菌株的目的基因的mRNA变化规律，从而研究长期保藏重组毕赤酵母菌株表达TAT-SOD的适合性及其条件优化。研究结果表明，摇瓶发酵的最佳条件下（YPDM培养基，体积分数1.0%诱导剂浓度，诱导温度30.0 ℃，初始pH值7.0），发酵液上清酶活水平为753 U/mL，是未优化初始酶活水平的5.1倍，其中pH值优化使TAT-SOD表达水平提高到原始值的3.4倍。7.0的初始诱导pH值的mRNA水平是对照组pH 4.0的3.5倍。这些结果说明，长期保藏的重组毕赤酵母菌株仍然适合表达TAT-SOD，发酵条件会对毕赤酵母的TAT-SOD表达造成显著影响，影响表达水平的最主要因素是目的蛋白质的mRNA水平变化。

Abstract:

TAT-SOD is a fusion protein of TAT protein transduction domain and SOD. The TAT-SOD recombinant Pichia pastoris constructed in 2007 was used as the original strain to study the effects of different temperature,initial pH value, and inducer concentration on the expression of TAT-SOD in shake flask fermentation. Spectrophotometric analysis, scavenging of superoxide free radical and SDS-PAGE were used to measure the concentration of microorganism, the concentration of target protein TAT-SOD and the enzymatic activity of SOD, respectively. The mRNA changes of the target gene in the expressed strain were analyzed by quantitative PCR, aiming to investigate the suitability and optimization of the expression of TAT-SOD in long-term preserved recombinant Pichia pastoris strain. The results indicted that YPDM medium, inducer concentration of 1.0% (V/V), induction temperature of 30 ℃, initial pH value of 7.0 were the optimal condition of shake flask fermentation, with an enzymatic activity level in fermentation supernatant of 753 U/mL which was 5.1 times of that in the initial conditions．When the initial pH was 7.0, the expression level improved to 3.4 times and the mRNA level was 3.5 times of that under the control pH at 4.0. These results indicate that long-term preserved strain of recombinant Pichia pastoris is still suitable for the expression of TAT-SOD. The fermentation conditions significantly impact the expression of TAT-SOD, and the most important factor affecting the expression level is the mRNA level changes of the target protein.