用GAP启动子在毕赤酵母中组成型表达重组 猕猴桃果胶甲酯酶抑制剂

目的构建组成型表达重组猕猴桃果胶甲酯酶抑制剂(kiwi pectin methylesterase inhibitor, kwPMEI)的毕赤酵母(P．pastoris)GS115工程菌株,探索碳源(葡萄糖、甘油、甲醇)对重组菌表达kwPMEI的影响,纯化kwPMEI并鉴定其对番茄果胶酶的抑制活性。方法应用PCR方法从P．pastoris GS115染色体中扩增了三磷酸甘油醛脱氢酶启动子(pGAP),以其取代诱导型表达载体pPIC9K-kwPMEI上的醇氧化酶启动子(pAOX1),构建了组成型表达载体pGAP9K-kwPMEI,并转化至GS115中。用Tricine-SDS-PAGE和Western blot分析目的蛋白表达情况,镍柱亲和层析纯化目的蛋白,并用凝胶扩散方法鉴定其抑制活性。结果重组毕赤酵母工程菌株成功组成型表达了kwPMEI,48h即达到最大表达水平,表达量约为66 mg/L。并且以甘油为碳源时kwPMEI表达量最高。成功分离纯化了kwPMEI,并经凝胶扩散方法检测表明其具有抑制活性。结论成功构建了组成型分泌表达kwPMEI的毕赤酵母菌株,为kwPMEI在果蔬汁中的进一步应用奠定了基础。

Constitutive Expression of kiwi Pectin Methylesterase Inhibitor inPichiapastorisUsing the GAP Promoter

Objectives To construct P. pastoris strains GS115 which can constitutively express recombinant kiwi pectin methylesterase inhibitor (kwPMEI) and to explore the effect of carbon source (glucose, glycerol and methanol) on the expression of kwPMEI, purify kwPMEI, and identify its inhibitory capacity to the tomato PME. Methods The GAP gene promoter was amplified from P. pastoris GS115 using PCR, and used to replace the AOX1 promoter (pAOX1) on pPIC9K-kwPMEI, resulting in plasmid pGAP9K-kwPMEI, which was subsequently transformed into P. pastoris GS115. KwPMEI was identified by Tricine-SDS-PAGE and Western blot, and purified by nickel affinity chromatography. The inhibitory capacity of kwPMEI was verified with a gel diffusion assay. Results The combinant P. pastoris strain constitutively expressed kwPMEI successfully, and the peak concentration of kwPMEI (66 mg/L) was obtained only after a 48-h fermentation. In addition, glycerol was the best carbon source for producing kwPMEI. Finally, KwPMEI was successfully purified, and it showed an inhibitory capacity by gel diffusion assay. Conclusion The P. pastoris strains constitutively expressing kwPMEI were constructed successfully, which built up the foundation for the further application of kwPMEI in juice industry.