Construction, Expression And Purification Of A RTargeted Toxin: Disintegrin_ADAM15-Adapter-Melittin

Cancer is one of the diseases that threaten seriously human lives and health. The anti-tumor technology has a very rapid development, but there has not been a common treatment for cancer so far. Nowadays, radiotherapy, chemotherapy and surgery are in common use. because of large usage ,lack of specific pharmacological activity and having serious side effects, the curative effect of has been at a low level all the time. So targeted cancer therapy is an important way to improve the therapeutic effect.Targeted drug for targeted therapy generally consists of three parts: a carrier, effector molecules(warheads) and adapter that link the carrier and warhead. An ideal targeted drug should have no systemic toxicity, which means that the carrier should have a specific orientation of the selected targets; warhead should have a strong ability of killing tumor cell; adapter should be relatively stable and must crack effectively when the drug reach the targets.Current targeted drug did not play the desired effect. There are many reasons. There are not strong target for some drugs. Some are lack of cracked adapter or adapters are unstable in the circulatory system, leading not to targeted drug effectively killing tumor cells and there being systemic toxicity.In order to solve these issues of targeted drugs in the practical application , we did the following study. First of all, we did a lot of work for the design of targeted drug.Integrin involved in almost every step of tumor cell metastasis and influence interactions of tumor cell and its microenvironment with the progress of the tumor.αvβ3 is one of the integrin family and positively correlated with metastasis of tumor cells, whileαvβ3 plays a key role in the process of tumor angiogenesis and highly expressed in tumor cells. These features ofαvβ3 make it an ideal target for cancer therapy. Consideringαvβ3 as the target, we designed the targeted drug DAM.αvβ3 combines with its specific ligand through the RGD sequence. The disintegrin domain of ADAM15 contains the RGD (Arg-Gly-Asp) sequence. We use part of the disintegrin of ADAM15 as a target-oriented carrier which combines with theαvβ3 of tumor cells abnormally expressed. So warheads will be anchored in the tumor cell surface making the re-targeted toxin concentrating in tumor cell to kill cancer cells, while other non-target organs drug concentration is low. So far this sequence has not been used as a carrier of targeted durg.Adapter determines the stability and target system toxicity of recombinant targeting toxin in large measure. Therefore, searching for adapter which is high stability; whose protease has higher cleavage activity in vivo, while the adapter is the best substrate of zhe protease is very important. Malignant cells excessively express urokinase-type plasminogen activator (u-PA), while u-PA receptor withαvβ3 form complex. Since these features of u-PA, we use a physiological substrate of u-PA as the adapter. When the target toxin combined withαvβ3, u-PA gets in close touch with the targeted toxin, thereby efficiently cuts target toxin and the warhead is released. The protease cutting adapter of anti-tumor targeted drug has been patented by us.In this study, we selected melittin, which effect on cell membrane as the warhead. melittin can be specifically transported to tumor cell surface by targeted carrier and rapidly released under the conditions of existence of u-PA to integrate into the tetramer. The tetramer kills tumor cell, avoiding its side effects when used alone. Based on the above design, we synthesized a re-targeted toxin, "disintegrinADAM15-adapter-melittin"named as DAM, We linked it to the prokaryotic expression vector pGEX-2T. Then a transformation was made to E.coli-BL21(DE3). the transformants were induced to express the target protein by IPTG. The product of expression was detected by SDS-PAGE, an evident band appeared at the predicted position(molecular weight 39 kD), indicated that the fusion gene had expressed in the E.coli cell. Useing glutathione Sepharose 4B affinity chromatography, we successfully purified soluble fusion protein GST-DAM. Then GST- DAM fusion protein was digested by thrombin to release the DAM monomer protein and DAM was purified by glutathione Sepharose 4B affinity chromatography with purity up to 97%. Determined by SDS-PAGE, the molecular weight of target band was about 13 KD as the same as the theoretical values, which indicated that digestion is successful.This study has explored four optimum conditions of expression: the densities of bacteria cells when the inducer is added to the medium; the concentrations of IPTG; the inductive durations and the temperature. From zhe experiments, we made a conclusion: the optimum density of bacteria cells when the inducer is added to the medium is 0.6～0.8 (OD600); the optimum concentration IPTG is about 1.0 mmol/L the optimum inductive duration is about 4 h; the optimum temperature is 25℃.The expression vector used was pGEX-2T which contain encoding glutathione S-transferase (GST) sequence in this study, that made expressed DAM protein in the form of fusion protein. Fusion protein made the solubitity of protein increase, the purification of the proteins easier under mild conditions. However, protein expressed in this study had also the presence of inclusion bodies, So urea was used to denature inclusion bodies, then inclusion bodies, was refolded by dialysis to make part of the inclusion bodies into soluble protein, which was purified useing NTA-Ni2+-Sepharose affinity chromatography.In this study, we used the targeted toxin to act on cultured lung cancer cells A549, that initially proved that expressed targeted toxin had the role of anti-tumor in vitro.