论文标题:毕赤酵母表达重组人胰激肽原酶
Recombinant Human Pancreatic Kallikrein Expressed in Yeast Pichia Pastoris
论文作者 袁新清
论文导师 陈劲春,论文学位 硕士,论文专业 生物化工
论文单位 北京化工大学,点击次数 83,论文页数 75页File Size1249k
2004-05-29论文网 http://www.lw23.com/lunwen_958853012/ 重组人胰激肽原酶; 毕赤酵母;表达;纯化;酶活
recombinant human pancreatic kallikrein; Pichia pastoris; expression; purification; enzyme activity
胰激肽原酶（pancreatic kallikrein，PK）作用于低分子量的激肽原释放出赖氨酰缓激肽 (又名胰激肽原)。激肽原酶和激肽系统（KKS）是维持血压平衡的一个重要组成部分，对血压、电解质平衡、炎症反应等生理或病理过程进行调控。因此，胰激肽原酶具有广泛的药用价值，除降血压之外，还可以用于治疗糖尿病肾病、改善肾脏功能，治疗心、脑血管疾病等。本文利用嗜甲醇毕赤酵母（Pichia pastoris）表达重组人胰激肽原酶（human pancreatic kallikrein，hPK）。首先将hPKcDNA克隆至毕赤酵母穿梭表达质粒pPIC9K构建重组质粒pPIC9K/hPK，经BglII线性化酶切，电穿孔将其整合到毕赤酵母GS115 (His-Mut+)染色体上，利用MM、MD平板筛选His+Muts型阳性克隆，含不同浓度G418-YPD平板筛选高拷贝转化子。共筛选到27株阳性克隆，其中3株是高拷贝。筛选到的高拷贝进行BMGY/BMMY摇瓶培养，诱导表达重组hPK。表达产物经SDS-PAGE凝胶电泳分析，重组目标蛋白质量分数达总蛋白的30％以上；相对分子质量为37 000，较理论设计值26 500大，由糖基化分析是因目标蛋白发生糖基化所致。 在基础盐培养基中进行摇瓶表达，初步优化了培养条件，在pH6.0、60％的原盐浓度条件下生长较好。透析脱盐后的发酵液经几种离子交换层析柱进行初步的分离纯化，比较发现弱阴离子DEAE Sepharose FF作用效果较好，目标蛋白获得选择性吸附洗脱，分离体系得到浓缩。纯化得重组hPK经Trypsin进行酶原活化后测定酶活，比活达5.6U/mg。动力学参数测定Km值为5.4×10-2mM，与文献值接近，进一步说明重组hPK在毕赤酵母中获得了正确的表达。初步建立了培养、分离重组hPK的工艺，为进一步的研究工作提供了参考。
Kallikreins are a group of serine proteases that are found in diverse tissues and biological fluids in human body. Pancreatic kallikrein has the ability to release efficiently a bioactive kinin from a kininogen. The kallikrein-kinin system is involved in the control of blood pressure, electrolyte balance, inflammation, and other diverse physiological processes. Thus, the pancreatic kallikrein has widely pharmaceutical action, including degrading the blood pressure and treating the diabetic nephropathy，cardiovascular ischemic and cerebrovascular complications. In the study, the human pancreatic kallikrein cDNA was synthesized and inserted into the plasmid pPIC9K to construct recombinant plasmid pPIC9K/hPK followed by linearizing by restriction endonuclease BglII. Then it was transformed into the host Pichia pastoris GS115 by eletroporation. On MM and MD plates, 27 His+Muts positive colonies had been screened. 3 colonies with high-copy purpose gene were gained by the G418 (geneticin) resistance screening. Among them, one colony was cultured in BMGY/BMMY flask for the expression of recombinant human pancreatic kallikrein in vitro. The main band of the fermentation supernatant on SDS-PAGE was about 37 000 and the expression level of purpose protein was about 30% of total soluble proteins by gel analysis. The expressed purpose protein was glycosylated confirmed by the glycosylation analysis.To further industrialized production, the base salt medium was used for expressing the recombinant hPK. Some cultivating condition such as pH, salt concentration and organic nitrogen source, was optimized. The dialysis deslating and ion exchange chromatography were performed as the primary purification methods. Comparing the adsorption-elution effect of several columns, the selectivity of weak anion exchange column, DEAE Sepharose FF, was better for the object protein hPK purification.The purified hPK enzyme activity was mensurated after prohPK was activated by trypsin. The hPK specific activity reached 5.6U/mg. By analysing of the kinetic parameter of recombinant hPK, the Michaelis contant is 5.4×10-2mM，near to the value reported by literature, which further confirmed that the recombinant hPK was exactly expressed in Pichia pastoris.
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