人源类溶菌酶蛋白6在毕赤酵母中的重组表达及活性分析

Expression of Human Lysozyme-like Protein 6 in Pichia pastoris and Analysis of Enzymatic Activity of the Protein

利用毕赤酵母(Pichia pastoris)重组表达人源类溶菌酶蛋白6(human lysozyme-like protein6,h Lyzl6),对其酶学性质进行分析。根据毕赤酵母密码子偏爱性设计并人工合成h Lyzl6基因,将其连接至含有乙醇氧化酶启动子(AOX1)的p PIC9K质粒构建重组表达载体p PIC9K-hlyzl6;重组表达载体经线性化后电转化入毕赤酵母GS115感受态细胞,经G418筛选获得高拷贝重组菌株后进行甲醇诱导表达。经甲醇诱导72 h后发酵液上清中酶活性达到最高值,发酵液上清经SDSPAGE检测在14.8 k Da处有重组h Lyzl6蛋白条带,分子量符合预期,通过甲壳素亲和层析可对其进行纯化;采用比浊法测定h Lyzl6酶学活性,结果表明h Lyzl6对溶壁微球菌(Micrococcus lysodeikticus)有较好的杀灭作用,最适反应温度为40℃,最适p H为5.5,其酶活力为54 700U/mg,Cu2+对其活性有明显抑制,EC50为30.2799 mg/L。采用基因工程方法首次在毕赤酵母GS115成功表达了重组h Lyzl6,证实其在体外具有杀菌活性,初步揭示h Lyzl6在男性生殖系统先天性免疫中发挥了一定作用,为进一步研究h Lyzl6的功能和应用开发奠定了基础。

The aim is to realize the recombinant expression of human lysozyme-like protein 6（ h Lyzl6） by the Pichia pastoris expression system and to investigate its enzymatic activity. The h Lyzl6 gene was synthesized chemically according to the codon usage preference of Pichia pastoris and cloned into the expression vector p PIC9 K. The recombinant plasmid was linearized and transformed into competent Pichia pastoris GS115. After screening by G418 plate,the yeast strain was cultured and induced with methanol. The recombinant protein was purified by chitin affinity chromatography and subjected to activity assay. The results showed that the lytic activity in the culture supernatant reached the peak after 72 h of induction and the existence of the recombinant protein was confirmed by SDS-PAGE. The recombinant h Lyzl6 exhibited bactericidal activity against Micrococcus lysodeikticus by using turbidimetric assay and had the activity of 54 700 U / mg. The optimum temperature and p H for h Lyzl6 were 40℃ and 5. 5 respectively and Cu2 ＋was found to have strong inhibition effect on h Lyzl6. The EC50 value was 30. 2799 mg / L. In conclusion,recombinant h Lyzl6 was expressed successfully by the Pichia pastoris strain GS115 and had in vitro bactericidal activity,which indicated that h Lyzl6 may play a role in innate immunity of the male reproductive system and laid a foundation for its functional research and application.