Expression Of CdMT1-MMP And Screening Of MT1-MMP Affity Peptides From Phage Display Libraries

Matrix metalloproteinases (MMPs) have been associated with cancer-cell invasion and metastasis, so they become the hotspots for the cancer research. Matrix metalloproteinases (MMPs) are a class of zinc-requiring extracellular endopeptidases that together can degrade all components of the extracellular matrix and basement membranes. The family of human matrix metalloproteinases (MMPs) currently comprises 25 zinc metalloproteinases. They are capable of hydrolyzing all known constituents of the extracellular matrix (ECM). According to their substrate structure, MMP family can be classified into eight distinct structural classes: five are secreted and three are membrane-type MMPs (MT-MMPs).The expression and activity of MMPs are increased in almost every type of human cancer, and this correlates with advanced tumor stage, increased invasion and metastasis, and shortened survival. Unlike classical oncogenes, MMPs are not upregulated by gene amplification or activating mutations. The only two reported genetic alterations in cancer cells are translocation of the MMP genes in neuroblastoma and amplification of the MMP gene. So, the increased MMP expression in tumors is probably due to transcriptional changes rather than genetic alterations. This might be the result of activation of oncogenes or loss of tumor suppressors;MMPs have been the focus of much anticancer research and clinical trials. Several agents have been developed that block the synthesis of MMPs, prevent them from interacting with the molecules that direct their activities to the cell surface or inhibit their enzymatic activity. Several unconventional MMP inhibitors are also being tested in clinical trials.MT1-MMP (MMP-14) was the first discovered MT-MMP. Consistent with other MMP family members, MT1-MMPs have a propeptide, catalytic domain, linker region, and a hemopexin domain. MT1-MMP demonstrated broad-spectrum proteolytic activity. MT1-MMP was found to hydrolyze type Ⅰ, Ⅱ, and Ⅲ collagen as well as gelatin, proteoglycan, fibronectin, vitronectin, and laminin. The characteristic 3/4 and 1/4 collagen fragments that are known to be generated by MMP cleavage of collagen were shown. Other physiological substrates that were later identified for MT1-MMP. Additionally, MT1-MMP is involved in processing the cell adhesion molecule, CD44, and the γ2 subunit of an ECM component, laminin-5. The soluble fragments generated by these specific cleavages may be involved in ligand binding, cell signaling, enzyme inhibition, etc.We have carried out these works:1. We have expressed the cdMTl-MMP in the constructed expression vector. Because of the E. coli expression system, it can rapidly express a plethora of proteins. Although it has the high expression level, the proteins aggregate inclusion bodies and loss activities. For this reason, we have to refolding the target proteins. Cells were induced by adding IPTG for four hours when the cell density reached an A600 of 0.4-0.5. Inclusion bodies were purified by chromatogram of an ion-exchange chromatography.2. Inclusion bodies were refolded by dialysis and gel filtration chromatography. After refolded, some proteins have activities. But the effects of refolding are not ideal.3. Catalytic domain of MT1-MMP enzymatic activity was analyzed by gelatin zymography and cleaving AAT.Then Km and Kcat/Km of cdMTl-MMP were determined.4. In order to screen the specific sequence binding or inhibiting with MT1-MMP, we applied the Phage Display Peptide Library Kit. At the beginning, we synthesized and purified a specific peptide corresponding to a unique insertion sequence in the cdMTl-MMP. Then, the peptide was used to screening target. After three and four rounds, individual clones are characterized by DNA sequencing. And we obtained the consensus sequences HXH.5. After three and four rounds, individual clones are characterized by ELISA. The KD values of several clones were determined.6. We also have to do many works, including the binging ability of peptide with cdMTl-MMP, inhibitory effect against cdMTl-MMP.