Expression And Purification Of CdMMP-26 Fused With Self-cleaving Aggregation Tag

The matrix metalloproteinases (MMPs) are a family of zinc-dependent proteinases involved in the degradation of the extracelluar matrix (ECM). The MMPs have been implicated in the processes of tumor angiogenesis, growth, invasion, and metastasis; and their expression and activation is increased in almost all human cancers compared with normal tissue.Matrix metalloproteinase 26/endometase/matrilysin-2 is a novel member of the matrixin family, was first cloned from human endometrial tumor cDNA. MMP-26 has specific biochemical characteristics and substrate specificity. It could promote human epithelial cancer cell invasion and metastsis. Hitherto, there is no structure information of MMP-26 reported in the literature.We have carried out these works:First, an open reading frame of cdMMP-26, the catalytic domain of MMP-26, was insert into the vector pET-ELP-INTEIN, which contains the coding region of ELP-INTEIN, resulting in the fusion expression of ELP-INTEIN-cdMMP-26 in Escherichia coli BLR(DE3) under control of a leaky T7 promoter at lower temperature.Second, for purification of cdMMP-26, we introduce a new method for non-chromatographic purification of recombinant proteins expressed in Escherichia coli. This method is based on a reversibly precipitating, self-cleaving purification tag. The tag is made up of two components: an elastin-like polypeptide (ELP), which reversibly self-associates in high-salt buffers at temperatures above 30℃; and an intein, which causes the ELP tag to self-cleave in response to a mild pH shift. Thus, a tripartite ELP-intein-cdMMP-26 precursor can be purified by cycles of salt addition, heating and centrifugation. Once purified, intein-mediated self-cleavage, followed by precipitation of the cleaved ELP tag, allows easy and effective isolation of the pure, native cdMMP-26 without the need for chromatographic separations. Recoveries of 10-15 mg of cleaved, native cdMMP-26 per liter of shake-flask culture have been achieved, typically in 8-24 h depending on specific process parameters.Third, activity of cdMMP-26 was measured by three methods. (1)Gelatin zymography (2)α1-Antitrysin (AAT), which can be efficiently cleaved by cdMMP-26(3)DQ gelatin, which is a quenched fluorescein conjugate substrate, can be efficiently digested by cdMMP-26 to yield highly fluorescent peptides. Then the increase in fluorescence was monitored with a fluorescence microplate reader. All the measurement was manifested that the purified cdMMP-26 was active. And we have lied the foundation of protein structure analysis of cdMMP-26.