Studies On Expression、 Purification And Anti-tumor Activity Of Recombinant Targeted Toxin DLM-S3

Cancer is a disease caused by abnormal proliferation of cells, causing serious harm tohuman health. The traditional treatments for cancer mainly are surgery, radiotherapy andchemotherapy. Cancer cells can metastasis through the blood and/or lymphatic, making itdifficult for surgery to remove the cancer cell completely; The use of radiotherapy hascertain limitations; Chemotherapeutic drugs have obvious side effects on normal tissue, dueto their lack of specific pharmacological activity, and cancer cells will get tolerance to thosedrugs after a long term using. With the development of tumor biology, mechanism for theoccurrence and development of tumor gradually get clear, people have changed the strategyand targeted therapy is becoming a hot research.Targeted drugs are mainly composed of three parts: targeted carrier, effector molecules(warhead) and linker. By binding with surface molecules of tumor cells specifically, targetedcarrier can deliver the effector molecules to tumor tissue selectively for playing killing effect,at the same time, having little effect to normal tissue. However there are some problems fortargeted drugs, for example: the non-specific binding between targeted carrier and tumor cell,instability of the linker, effector molecules’spatial conformation changed and so on. Inresponse to these issues, we designed a targeted toxin which has high binding specificity,low side effects. It can release the effector molecules efficiently through a cleavable linker attumor, playing the cytotoxic function.The integrins are a class of cell surface adhesion molecules, involved in the process oftumor occurrence and development. The member αvβ3integrin is highly expressed in thesurface of many tumors and in the endothelial cells of tumor angiogenesis also highlyexpress. But in mature endothelial system and most normal tissues, αvβ3is not expressed orlow expression. These features make it an ideal target for cancer therapy. In this study, weuse the disintegrin as targeted carrier which can bind with αvβ3specifically. The recognitionsite of αvβ3to its ligand protein is RGD sequences. We use the Ussuri venom disintegrin ascarrier of targeted toxin, the ability of which binding to αvβ3is2000times higher than theRGD. This efficient and specific binding can transport the effector molecules to tumor cellsurface. Because of low concentration in normal tissues, this reduces the side effects.We choose melittin as effector molecule, which is comprised by26amino acids. At low concentration and low ionic strength melittin exists in the random coiled structure, but athigher concentrations and higher ionic strength melittin is able to assemble itself intotetrameric and amphipathic structures. This amphipathic property decides that melittin canbe dissolved in water and bound with membrane naturally. After be transported to the surfaceof tumor cells, it can destroy the integrity of cell membrane and make the cell content to leakout, and cells death.uPA is a serine protease which is highly expressed in many tumors. Its main function isinvolved in the decomposition and regeneration of basement membrane, which is closelyrelated to the metastasis of tumor. The uPAR and integrin αvβ3can form a complex bybinding with vitronectin together. The activity of uPA strengthens after binding with itsreceptor uPAR. So the uPA is enriched at the tumor receptor αvβ3. We inserted an uPAcleave site between the carrier and the warhead, when the targeted toxin combine with αvβ3,the tumor expressed uPA would cleavage the linker and release the warheads to kill tumorcells. The uPA cleave site LGGSGRSAV we choosed come from the best recognitionsequence, which is5000times higher than its physiological substrate sequence PGRVV.In summary, we used the snake disintegrin as targeted carrier, melittin as effectormolecules, inserted a linker peptide sequence SGGGSGGGSGGGS and uPA cleave sitebetween them to construct recombinant targeted toxin DLM-S3.In this study, pichia pastoris expression vector pPIC9K/DLM-S3was constructed. ThesacⅠlinearized plasmid pPIC9K/DLM-S3was transformed to strain GS115viaelectroporation. Transformants were screened by histidine defect, and then extracted thegenomic DNA for PCR identification. Selected high copy number transformants with highG418resistant. Induced with1%methanol for96h, expression level reached the peak.15%SDS-PAGE identified the supernatant of fermentation, there was a specific band near15kDa,showing that DLM-S3successfully secretory expressed by pichia pastoris.The high express level strain was screened through SDS-PAGE. Inducing for96h,collected the fermentation supernatant, concentrated it with ammonium sulfate, thendesalinated through dialysis. We used Ni-Sepharose6FF at pH7.6to purify the DLM-S3,200mM imidazole could elute the protein.15%SDS-PAGE showed a single band about15kDa, the purity was more than90%through grayscale scanning.The effect of DLM-S3on the proliferation of Hela, A549and MCF-7was analyzed byMTT assay,293cell as normal control. The result showed that low concentration had aninhibition on tumor cells’ growth, in dose-dependent manner. The IC50was5.09μg/ml, 5.48μg/ml and3.60μg/ml respectively. DLM-S3also could kill the tumor cell directly,causing membrane damaged, cell died. There was no apparent effect on293cell and theinnibition of16μg/ml DLM-S3to293cell is merely7.5%. Hemolysis experiment resultsshowed that DLM-S3had little hemolysis.We observed the influence of DLM-S3on theultrastructure of MCF-7by transmission electron microscope: membrance rupture,cytoplasmic vacuolization, organelles disappearing, showing necrotic morphology.This study constructed DLM-S3eukaryotic expression system and purified byNi-sepharose6FF affinity chromatography. In vitro experiment showed significant killingeffect on tumor cells.