Studies On Expression,Purification And Anti-tumor Activity Of RTargeted Toxin DL_S2M

Tumor is a disease which threatens seriously human health. At present the treatmenteffect of the traditional treatment method is not satisfactory. As a result of deep research oftumor, the tumor targeted drugs is becoming a trend of tumor treatment. The recombinanttargeted drugs generally consist of three parts: targeted carrier, effector molecule and linkerthat link the targeted carrier and effector molecule. The effector molecule can be transportedto target site by tumor-specific carrier. Linker can be cleaved efficiently at target site and theeffector molecule will be released to kill tumor. This study design a recombinant targetedtoxin that has highly target activity and can release the effector molecule efficiently.Integrin is a class of cell surface receptor family, involved in the process of tumorformation, growth, invasion and metastasis. The member αvβ3integrin is highly expressedin the surface of many tumor cells and in the neovascular endothelial cells of tumorangiogenesis. But in mature endothelial system and the majority of normal organs, αvβ3isnot expressed or low expressed. RGD（Arg-Gly-Asp） is the ligand recognition site of αvβ3.RGD is a extremely promising targeted carrier. In this study, the snake disintegrin containinga RGD sequence was used as the carrier of recombinant targeted toxin. The ability ofdisintrgrin binding to αvβ3is more than1000times of the RGD.In this study, the effector molecule we choose is melittin. Melittin is a2840Da smallmolecule which consisted of26amino acids. At low concentrations melittin exists in therandom coiled structure, but at high concentrations and high ionic strength melittin is able toassemble itself into tetrameric and amphipathic structures. Melittin can destroy the cellmembrane, then the cell contents leak out, and finally the cell dies.It is very important to choose a appropriate linker that can connect carrier and effectormolecule, because linker directly affects the stability of the recombinant targeted toxin andrelease of efector molecules. uPA is a serine protease which is highly expressed in manytumor cells. The activated uPA can combine with uPAR on the surface of tumor cell andcombine with αvβ3to form a complex. So the uPA is enriched at the tumor receptor αvβ3. Ifan uPA cleave site is used to be the linker, the uPA that the tumor cells expressed cancleavage the linker and release the effector molecules to kill the tumor. The physiologicaltarget sequence of the uPA is PGRVV. Studies has shown that the cutting efficiency of different target sequence of uPA are different. The efficiency to LGGSGRSV is as much asnearly1000times than that to PGRVV.As suggested above, in this study we used the snake disintegrin as the targeted carrier,melittin as the effector melocule and uPA cleavage short peptide LGGSGRSV as the linkerconstructed the recombinant targeted toxin DL_S2M. meanwhile we changed R at the left sideof LGGSGGSV to G by mutation, so LGGSGGSV lost uPA cleavage site and can not becleaved by uPA. We used this mutated sequence as the linker and constructed recombinanttargeted toxin DL_S4M, and used it as a control.This study prepared DL_S2M and conducted preliminarily research on the antitumoractivity of DL_S2M in the following aspects:1) Expression of DL_S2M、DL_S4M in Pichia pastorisPichia pastoris expression vector pPIC9K/DL_S2M and pPIC9K/DL_S4M wereconstructed,6×HIS were added to the N-terminal of DL_S2M and DL_S4M to facilitatedownstream purification. The SacⅠlinearized plasmids pPIC9K/DL_S2M and pPIC9K/DL_S4M were transformed to strain GS115via electroporation. Positive transormants wereselected with high G418resistant, induced with0.5%menthanol to express DL_S2M andDL_S4M. The expression products in the supernatant of fermentation was identified bySDS-PAGE. Strains with highly expressed level were screened.2) Purification of DL_S2M and DL_S4MStrains expressed DL_S2M and DL_S4M were conducted to fermentation by inducer of0.5%methanol for96h. Nickel chelating affinity chromatography was used to obtain therecombinant products, the purified products were then analyzed by SDS-PAGE. The puritiesare above90%.3) Activity assay of purified DL_S2M and DL_S4MWe observed that the lethal effects of DL_S2M to the A549and MCF-7cell lines areobvious, but the lethal effect of DL_S4M to these two cell lines are not obvious. This revealthat the linker of recombinant targeted toxin DL_S2M can be cleaved, and only after it iscleaved, the DL_S2M can play its cytotoxic effect.To verify the activity of Purified DL_S2M, growth inhibition assays on the tumor cellsA549and MCF-7cell line were conducted. The results show that the lethal effects of DL_S2Mto these two tumor cell lines are obvious.Concentrations of2μg/ml,4μg/ml,8μg/ml,16μg/ml of DL_S2M were incubated with293cells for48h, the results shows that DL_S2M almost has no cytotoxicity, that mean the DL_S2M has low cytotoxicit to normal tissues.In summary, this study constructed Pichia pastoris eukaryotic expression vector, andscreened the DL_S2M and DL_S4MP. Pastoris engineering strains with highly and steadyexpression level. The recombinant protein DL_S2M and DL_S4Mwere purified by Nickelchelating affinity chromatography. The lethal effect of DL_S2M on tumors were observed. Theaccomplishment of this study provides a new way to the research on tumor targeted drugsand have important scientific and practical significance.