Expression Of Matrix Metalloproteinase 26 In Prokaryotic And Eukarytoic Cells

Matrix metalloproteinase which is one of the most important enzymes that degrades the extracellular matrix is the generic name of endopeptidase secreted by cells sequestrated by Zn2+. They are widely distributed in animal and plant sector, almost degrade all the composition of biological elements in vivo extracellular environment matrix. In normal and pathological conditions, MMPs have a broad role in protein hydrolysis. In tumor generation and development, MMPs often over-express, and degradate basement membrane and extracellular matrix, promot tumor invasion, metastasis and angiogenesis. Therefore, MMPs as a therapeutic target, designing and searching for the antagonist drug, especially selectivity inhibitor as drugs, has become the hot spot in recent years. MMP-26 is the smallest matrix metalloproteinases, which has a special structure and activation mechanism. The expression in normal tissue has shown signs of cyclicity, but in tumor tissues the widespread and a wide range existence is very different, thus it becomes the hot topic in recent years. However, so far, its activation mechanism is not yet clear; protein structure has not yet been resolved. The current study of MMP-26 just stays in the study of its expression in a variety of tumors, but not its manual expression system. About its expression in vivo, all kinds of literature and research have not exactly the same results and inferences. In order to study the protein expression and activation form, we used the prokaryotic and eukaryotic expression system, trying to study the expression and the secretory pathway, laying the foundation for further study its structure and the activation mechanism.1. MMP-26 protein in prokaryotic expression, purification and refolding studies The best induction condition was IPTG concentration of 1mM for 4h when the cell density of OD595 between 0.5 ~ 0.6. The inclusion bodies were obtained after washing and dissolved with 8M urea buffer overnight and MMP-26 protein was a sigle strip on the SDS-PAGE after purified by the anion-exchange chromatography. Protein concentration should be diluted to 100mg/ml below. Dialysis refolding method was used stepping every two hours, and completed the refolding work within 12h. High activity MMP-26 protein was obtained, and the detection by Western- blotting revealed that a total of at least four activated form, with molecular weight, respectively: 17KD, 19.4KD, 22.3KD and 24KD. Among them, the strip with molecular weight of 19.4KD was the the main activation form, with the splice site at 90THE.2. Eukaryotic plasmid construction of MMP-26 full-length gene and stability transfection in COS-7 cell line, screening and identification positive clones.(1). Plasmid constructionFirstly, full-length MMP-26 gene was cloned to plasmid vector PCS4 making MMP-26 tightly connect to three repeated MYC tag sequence in the C-terminal. In order to obtain resistant labels in the vector for stable transfection, connect MMP26-MYC to the pcDNA3.1 plasmid.(2).Transfection of MMP-26 geneThe pcDNA-3.1 plasmid carrying full-length MMP-26 gene was transfected into COS-7 cells through the PEI Act. After G418 selection, choose a single cell clone for expansion of culture, six resistant clones within the pcDNA-3.1-MMP-26-MYC transfection group were obtained and identification method as below:The results of Western-blotting displayed MMP-26 protein expressed in two drug-resistant clones 4 and 8. RT-PCR detection of MMP-26mRNA in both positive clones was positive. Cell immunofluorescent staining useed anti-MMP-26 polyclonal antibody and FITC labeled secondary antibody to detect MMP-26 protein expression in cells and positioning. The results showed that fluorescence intensity increased in the cell cytoplasm transfected with MMP-26 gene, and it was strongly positive, distributed in the cytoplasm of transfected cells.(3). The impact of biological characteristics of COS-7 cells with MMP-26 protein expressed After the successful transfection of MMP-26, there were many cells with big size and the phenomenon of irregular edge. Although cultured cells were monoclonal, there were obvious differences in cell morphology, and the pcDNA-3.1 transfected cells and COS-7 cells had no such change. Transfected cells changes in the structure as the following:(1) Split-phase cells increased significantly, and a lot of mitotic cells could be saw; (2) a marked increase in cell death and proliferation to accelerate.In this paper, prokaryotic and eukaryotic MMP-26 proteins have been obtained. We acquired highly active MMP26 protein in prokaryotic and cell lines stable expressed eukaryotic MMP26 protein. RT-PCR, cell immunofluorescence and Western-blotting analysis revealed a preliminary comparative study of its forms of expression, self-activated form, path of secretion and the presumption the processing site. To study the role of MMP-26 in the normal tissues as well as the function and significance in the development of malignant tumors provides a model and also for further study of MMP-26 activation mechanism in vivo, thereby laid the foundation for target treatment of tumor.