Separation Of Heparanase Gene, Protein And Its Interacting Protein Screening

Metastasis and recurrence have been believed to be the major cause for cancer treatment failure. The process of tumor metastasis is highly complicated and consists of a series of sequential, interrelated steps, briefly including detaching from the primary tumor, local invasing into the host stroma and penetrating the lymphatic or vascular channels, surviving from immune defenses, vascularization and establishing a micrometastasis on the receptive organs.The key factors for protecting tumor metastasis include intact extracellular matrix (ECM) and basement membranes (BM). Heparan sulfate proteoglycan (HSPG) is the major molecular constituents of ECM and BM, its breakdown will make it easier for tumor to occur invasion and metastasis.Heparanase is a p-D-endoglucuronidase that cleaves heparan sulfate (HS) from HSPG and degrades them to small oligosaccharides ranging from 10 to 20 disaccarides. It has been known for many years that the metastatic potential of tumour cells is related to their heparanase content. This observation has been confirmed recently by the finding that heparanase is highly expressed in various human tumors and closely related with invasion, metastasis and poor prognosis. Further support for the role of heparanase in tumor metastasis comes from transfection studies where the metastatic ability of melanoma cell line is substantially enhanced when they are stably transfected with the heparanase gene. Metastatic tumour cells are more active in expressing heparanase, the enzyme can not only breach ECM and BM barriers but also liberate and regulate a large number HS-binding growth factors from ECM depots and make them available for angiogenesis in tumor metastasis.Clearly heparanase presents a new promising target for antimetastatic drug development. It is much desirable to develop a new therapeutic method for tumor metastasis based on the behavior studies for heparanase, including its structure and function. However, such kinds of study just begin, although the full encoding gene for this enzyme has been reported in western country, the information is very limited in expression regulation and biological function for heparanase. To furthercharacterize the alterations of heparanase expression in China tumor samples and its expression reguLation is very important for designing anti-metastasis drugs from heparanase. Thus, the present study was designed to establish the full encoding gene and protein expression vector for heparanase and to determine the expression regulation pathway for heparanase with cell culture and molecular methods. Furthermore, the protein expression in mammalian cells and signal peptide influence on post-translation and activation for heparanase were determined. The co-acting protein of heparanase was preyed by the two hybrid screening approach in yeast for exploring its actual process in vivo.Materials and Methods:1. Heparanase gene isolation and ampliation: Extracting the total mRNA by SV Total RNA Isolation System kit from the HepG2 cells which were harvested in 80% confluence and synthesize the cDNA by ImProm-IITM Reverse Transcription System kit. The PCR reaction was performed by using primers corresponding to the full heparanase encoding gene and the cDNA template. The PCR products were cloned in pGEM-T vector for sequencing.2. Heparanase expression in mammalian cells: The subcloning technique was used to clone the full heparanase gene and the gene lacking its signal peptide region into the pcDNA4/Myc-His. The two constructed eukarytic expression vector pcDNA4/Myc-His full HPA and pcDNA4/Myc-His part HPA were transfected into cos-7 cells and CHO cells by lipofectine method. Western blot analysis was applied to detect the heparanase expression in both cells. The permanently expression cell lines were screened by selection reagent, zeocin.3. Heparanase expression in E.Coli.: The part sequences of heparanase 8KD and 50KD subunit were subcloned into the vector pGEX-2TK to constructe respectively the two GST-fusion expression vector pGEX-2TK-8KD...