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-2 C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IgG κ链和人基质金属蛋白酶-9(matrix metalloproteinase 9, 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 cell-based 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 supernatants 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 IgGκ 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 protein from culture media, resulted in detection of proteins with the predicted molecular mass, which indicate that all of the signal sequences could direct PEX secretion successfully. The MMP-9 signal peptide seems to be superior to the signal peptides from IgG and rGH both in terms of extracellular yield and in terms of secretion efficiency. Thus, expression of pM9PEX construct resulted in higher yields of extracellular PEX and the majority of the produced PEX was secreted and not trapped intracellularly. To examine whether the observed difference in secretion yields is promoted at the transcriptional level, a RT-PCR analysis was performed at 6 h after transfection. The presence of mRNA transcripts of PEX was observed in all the DNA constructs. Moreover, semiquantitative reverse transcription (RT-PCR) results show that there were no significant differences in the expression levels of PEX among the constructs at 6 h after transfection. Though there was no difference in the expression levels of PEX at an early time point after transfection, the presence of an ER-targeting signal peptide sequence in the expression vector affected the trafficking of expressed proteins in the cells. Hence, the described difference in exported yields is probably promoted at the secretion level, rather than at the transcriptional level. Chick chorioallantoic membrane (CAM) bioassay show that the PEX protein purified from cell culture had biological activity to inhibit the angiogenesis. The MMP-9’s signal peptide is used for the first time as leader sequence for secretion of foreign proteins. Our results revealed that higher amounts of secreted PEX were obtained when vectors containing MMP-9 signal peptide were used and it is also indicated that MMP-9 signal sequence could be effective on promoting the secretion of other heterologous proteins in eukaryotic cells.
宋琳,张志谦. MMP-9信号肽高效诱导PEX重组蛋白在COS7细胞中分泌表达[J]. 中国生物工程杂志, 2007, 27(5): 1-5.
. Efficient Secretion of Recombinant PEX in COS7 Cells. China Biotechnology, 2007, 27(5): 1-5.
https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2007/V27/I5/1
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