Abstract

Azurin, which is a bacterial secondary metabolite has been attracted as a potential anticancer agent in recent years because induced death of cancer cells and inhibited their growth. In this study, the production of azurin under the control of the alcohol oxidase promoter which is frequently used in the Pichia pastoris expression system was performed. The azurin gene amplified from Pseudomonas aeruginosa genomic DNA and inserted into the pPICZαA was cloned in Escherichia coli cells. Then, a linearized recombinant vector was transferred to the P. pastoris X-33 cells. Antibiotic resistance test and colony PCR were performed for the selection of multicopy transformants. Protein expression capacities of selected transformants were compared at the end of 48 h incubation. Both extracellular and intracellular protein expressions were observed in all of them by Western blot analysis. The relative expression levels of both intracellular and extracellular protein that belongs to the first clone were higher than the others. On the other hand, it was seen that the 4th clone had the highest protein secretion ability. The molecular mass of the extracellular azurin protein which is produced by recombinant clones was found to be about 20 kDa. This is the first report on azurin expression in P. pastoris.

摘要

近年来，由于细菌诱导的死亡并抑制了癌细胞的生长，天蓝色素是一种细菌的次生代谢产物，已被吸引为潜在的抗癌药物。在该研究中，在巴斯德毕赤酵母表达系统中经常使用的醇氧化酶启动子的控制下进行了天青蛋白的生产。从铜绿假单胞菌基因组DNA扩增并插入pPICZαA的天青蛋白基因被克隆到大肠杆菌细胞中。然后，将线性化的重组载体转移至巴斯德毕赤酵母X-33细胞。进行抗生素抗性测试和菌落PCR以选择多拷贝转化体。在48小时孵育结束时比较所选转化体的蛋白质表达能力。通过Western印迹分析在所有细胞中均观察到细胞外和细胞内蛋白表达。属于第一个克隆的细胞内和细胞外蛋白的相对表达水平均高于其他克隆。另一方面，可以看出第四克隆具有最高的蛋白质分泌能力。发现重组克隆产生的细胞外天青蛋白的分子量约为20kDa。这是关于巴斯德毕赤酵母中天青蛋白表达的首次报道。