构建一种表达人溶菌酶的游离型毕赤酵母工程菌。巴斯德毕赤酵母表达质粒多以整合型质粒为主,整合型毕赤酵母表达外源基因时,外源蛋白的表达量会随着基因整合拷贝数的增加而增加,而对于毕赤酵母而言,稳定的游离型载体更有利于进行外源基因突变文库的构建及高通量筛选。该文将来自乳酸克鲁维酵母中的自主复制序列(pARS)与酵母表达载体pPICZαA连接,得到游离型表达载体pPICZαA-pARS。将优化后的人溶菌酶基因hLYZ连接至该载体中,构建重组表达载体pPICZαA-hLYZ-pARS。重组载体经电转化以游离形式转入毕赤酵母菌株X33中。摇瓶发酵结果显示诱导72 h后,发酵上清液酶活性为340 U/mL。利用镍亲和层析从发酵上清液中纯化人溶菌酶,二喹啉甲酸(bicinchoninic acid,BCA)法测定后纯化后的蛋白质量浓度为0.954 mg/mL。该研究成功构建了分泌表达具有生物活性的人溶菌酶的游离型毕赤酵母工程菌,为日后利用现代生物技术突变获得更高抗菌活性、更广抗菌谱人溶菌酶的研究奠定了基础。
In this study, an episomal Pichia pastoris expression vector was constructed for the recombinant expression of human lysozyme. The commonly used expression vectors in P. pastoris are mainly integrative ones. When the integrative plasmid repeatedly integrates into the chromosome of P. pastoris by homologous recombination, the expression level of heterologous proteins will increase with the increasing copy number of the gene. However, in P. pastoris, stable episomal expression vectors benefit the construction of mutation libraries of heterologous genes and subsequent high-throughput screening. In this paper, the autonomously replicating sequence (pARS) from Kluyveromyces lactis was inserted into the yeast expression vector pPICZαA to obtain the episomal expression vector pPICZαA-pARS. The optimized gene sequence hLYZ was cloned and ligated to the episomal expression vector to construct the recombinant expression vector pPICZαA-hLYZ-pARS, which was then transformed into P. pastoris strain X33 by electroporation. After 72 h of cultivation and induction by methanol in shake-flask, the enzyme activity was 340 U/mL in the supernatant. Then the human lysozyme was purified by nickel affinity chromatography, and the concentration of purified human lysozyme was 0.954 mg/mL determined by BCA. In this study, a P. pastoris engineering strain harboring an episomal vector to express and secrete human lysozyme had been successfully constructed, which laid a foundation for the further studies on using modern mutagenesis technology to screen for human lysozyme mutant with higher antibacterial activity and wider antibacterial spectrum.
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