MMP-9信号肽高效诱导PEX重组蛋白在COS7细胞中分泌表达  

Efficient Secretion of Recombinant PEX in COS7 Cells Directed by MMP-9 Signal Peptide

作　　者：宋琳[1] 张志谦[1] 

机构地区：[1]北京大学临床肿瘤学院/北京市肿瘤防治研究所,北京100036

出　　处：《中国生物工程杂志》2007年第5期1-5,共5页China Biotechnology

基　　金：国家自然科学基金资助项目(30270658);北京市自然科学基金资助项目(7002009)

摘　　要：为了便于收集和纯化,重组蛋白常需要引导至真核细胞外。蛋白能否分泌主要取决于其是否含有信号肽,由于不同信号肽诱导蛋白分泌的效率不同,高效信号肽的筛选已成为生物工程领域提高重组蛋白产量的重要策略之一。为了筛选诱导MMP-2C末端PEX在COS7细胞中高效分泌表达的信号肽,在PEX的N末端分别融合大鼠生长激素(rGH)、小鼠IgGκ链和人基质金属蛋白酶-9(matrix metalloproteinase9,MMP-9)的信号肽并比较三种信号肽引导PEX分泌表达的效率。Western免疫印迹和ELISA蛋白定量检测表明MMP-9的信号肽引导PEX蛋白分泌的效率约为其它两种信号肽的两倍。利用Ni-NTA亲和柱对细胞培养基中的PEX进行纯化,蛋白产量约为1mg/L,纯化的PEX重组蛋白具有抑制鸡尿囊膜(chorioallantoic membrane,CAM)血管发生的作用。以上结果提示MMP-9的信号肽有效诱导具有生物活性的PEX重组蛋白在COS7细胞中分泌表达。With the rapidly development of the biotechnology industry, large quantities of recombinant proteins are needed for specific therapeutic and diagnostic applications. Bacterial cells are most often used for the production of recombinant proteins. However, recombinant proteins expressed in the cytoplasm of bacteria are often misfolded as insoluble inclusion bodies and therefore inactive. To circumvent this problem, several eukaryotic expression systems have also been developed over the years, ranging from yeast to mammalian cellbased technologies. For many mammalian proteins, especially those secreted and modified posttranslationally, a more compatible expression system is highly desirable because proper folding or modification can only be provided with closely related cells, i. e. , mammalian cells. Large scale transient transfection of mammalian cells is a recent and powerful technology for the fast production of milligram amounts of recombinant proteins. Transient expression by means of extrachromosomal replication in COS cells is frequently used to check the functional integrity of genes/plasmids and to produce small quantities of cell supematants containing the protein of interest.As it is allowed for easy and efficient purification, many recombinant proteins used for therapeutic and structural studies are naturally secreted or engineered to be secreted. The use of a proper signal peptide is one of the major determinants for the efficient secretion of heterologous proteins from mammalian cells. The noncatalytic C-terminal hemopexin-like domain of MMP-2, PEX, can block angiogenesis and tumor growth in vivo. Large quantities of biochemically active recombinant PEX are required for the study of their functions and biochemical properties, as well as for their industrial applications. For this purpose, the rat growth hormone, mouse IgGK chain and MMP-9 signal peptides were used for expression of PEX in COS7 cells, and their secretion efficiencies were compared by Western blotting and ELISA. Western-blotting of PEX

关 键 词：基质金属蛋白酶-9 信号肽 分泌表达载体 PEX 

分 类 号：Q786[生物学—分子生物学]