Expression And Identification Of Endostation Fusion Protein In Pichia Pastoris

O' Reilly et al found a new anti-angiogenesis factor called endostatin was isolated from cultures of mouse haemangioendothelioma(EOMA) in 1997. Endostatin can inhibit the proliferation of endothelial cells, and then inhibit the growth of new blood vessels. The growth and metastases of tumors depend on neovascularization, thus endostatin become a new strategy for tumor therapy. A new commercial anticancer drug named Endostar only has the short half-life of about ten hours. So this drug needs to be injected to patients one time a day and it increases patients' burden. Therefore, it is important to develop some kinds of long-acting endostatin agents. The aim of this research program is to construct endostain-HSA fusion gene and express the fusion protein to prolong the half-life of endostatin, which lay the foundation for the development of long-acting endostain drug.1. The prediction of EndoAlbu's spacial structureTo prolong Endostatin's half-life, linker(Gly4Ser)3 , which is rich in glycine and serine, is used to connect Endostatin and HSA to form a fusion protein. Three similar domains make up of HSA protein, every domain contains two subdomains which form a cylindraceous structure. Most of these hydrophobic amino acids locate in the inside chamber of the cylindraceous structure and hydrophobic chambers are produced. In order to verify whether this special structure of the HSA protein will affect functional sites of Endostatin, we predicted the structure of fusion protein. The result indicated that the Endostatin exhibit the independent structure in this fusion protein.2. The construction of EndoAlbu fusion geneThe EndoAlbu fusion gene was constructed by overlap extension PCR method, and then cloned into the pGEM-T vector. The DNA sequencing of fusion gene showed that the human endostatin gene and human serum albumin gene were exactly identical to AJ 494837 and CR 607928 sequence in GenBank of NCBI respectively. 3. The expression, identification and purification of EndoAlbu fusion proteinEndoAlbu gene with correct DNA sequence was cloned into pPIC9 vector to form recombinant expression vector pPIC9-EndoAlbu. Then the correct expression vector was linearized and transformed into GS115. High-yield expression strains were screened by in situ double-film method and extensively cultured after identification. Fusion protein was purified by the methods of ammonium sulfate precipitation and Blue Sepharose 6B Fast Flow affinity chromatography from the yeast culture supertant.4. Bioactiveity detection of EndoAlbu fusion proteinThe purified EndoAlbu was detected by MTT method and results showed that it could obviously inhibit the proliferation of human vein endothelial cells in a dose-dependent manner and the IC50 was estimated to be 8.4μg/mL. Purified EndoAlbu could also inhibit the formation of new blood vessels in chicken chorioallantoic membrane(CAM) experiment.5. ConclusionOn the base of the prediction of EndoAlbu's spacial structure, the EndoAlbu fusion gene was constructed, and then the fusion protein was successfully expressed and purified. The result indicates that EndoAlbu fusion protein has favorable bioactivity, which provides theoretical basis for the preparation of long-acting endostatin.