Cloning And Expression Of Recombinant Human Heparanase Gene And Research Of Characterization Of Heparanase

Metastasis and recurrence have been believed to be the major cause for cancer treatment failure. Heparan sulfate proteoglycan (HSPG) is the major molecular constituents of extracellular matrix and basement membranes, its breakdown will make it easier for tumor to occur invasion and metastasis. Heparanase (HPA) is a p-D-endoglucuronidase that cleaves HSPG. It has been known that the metastatic potential of tumour cells is related to their heparanase content. 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 studies for heparanase, including its structure and function.The heparanase cDNA encodes for a pro-preHPA., the signal peptide is cleavaged after secretion. The preHPA undergoes a proteolytic cleavage that is likely to occur at two potential cleavage sites, yielding an 8 kDa polypeptide and a 43 kDa polypeptide. It has been hypothesized that heterodimer formation between the 8 kDa and 43 kDa heparanase subunit is important for heparanase enzymatic activity.Aim to reserch the relationship between the structure of HPA and its activity and the function of HPA in the process of tumor metastasis, it's needed to gain therecombination HPA. For this purpose, two vectors for intracellular expression were also constructed and transformed into E.coli . Four vectors for secreted expression in Pichia in different plasmid were constructed and transformed into pichia pastoris GS115 and X33. The preHPA and the 43 kDa subunit protein of heparanase fusing with His X 6 tag protein was successfully expressed in E.coli.we got purified HPA and 43 kDa fused with His X 6 tag by seriesly purification, which be used to prepare the Anti-HPA PcAb and Anti-HPA-subunit PcAb. By means of FIA, we identify the relationship of the expression level of HPA and serval kinds of inviding cancer cells . We show that fibroblasts are capable of activate extracellular HPA. We demonstrate specific association between the heparanase subunits by means of GST pull-down experiments.The present results was fundamental for further characterization on heparanase and its structure, biological function in tumor metastasis and anti-metastatic drug development.