| Angiogenesis is associated with several pathological disorders. Tumor growth and metastasis are dependent on angiogenesis. Components of vascular basement membrane are involved in regulating angiogenesis. Tumstatin (the NC1 domain of 3 chain of typeⅣcollagen) was identified as possessing anti-angiogenic activity. the anti-angiogenic activity of tumstatin was localized to the 54-132-amino acid Tum-5 domain, the activity mediated byαvβ3 integrin interaction in an RGD-independent manner,and the anti-angiogenic activity of Tum-5 is same as Tumstatin.Cyclic peptide NGR (CNGRCVSGCAGRC), homing specifically to tumor vascular was screened by in vivo phage display. The receptor of NGR has demonstrated to be aminopeptidase N (CD13) which homes the tumor blood vessel endothelium cells specifically. The CD13/APN has been approved to mediate migration and invision of endothelial cells in the process of angiogenesis. A peptide containing the NGR motif, the main domain of CNGRCVSGCAGRC, has been coupled to different antitumor drugs, such as doxorubicin, cytokines and proapoptotic peptides, and yielded compounds with increased efficacy against tumors and lowered toxicity to normal tissues in mice.To enhance the antitumor effects, cyclic peptide NGR was bound to the C terminus of Tum-5 in this experiment. We expected that the yielding fusion protein could either bind with the CD13/APN homing specifically on tumor endothelial cells or inhibit angiogenesis that mediated with CD13/APN.For the reason that there was no commercial antibody available, rabbit polyclonal antibody against Tum-5 was prepared firstly for later works such as western blot and Elisa. Tum-5 protein expressed in E.coli was used in this step.After transfected with recombinant plasmids of pcDNA-tum-5-NGR or pcDNA-tum-5, the stable CHO transfectants were screened by G418 for the selection of those secreting recombinant protein. Moreover, the growth rate of stable transfectants was not affected by the secreted protein. The in vitro anti-angiogenic activity of recombinant protein was proven to inhibit the proliferation, tube formation, migration of endothelial cells.Expression of recombinant protein was detected using RT-PCR after muscle injection.in vivo. To investigate the inhibition effect on tumor growth, recombinant plasmids was injected into BALB/c mice bearing S180 tumors. A significant anti-tumor effect of pcDNA-tum-5 and pcDNA-tum-5-NGR was observed. The weight of tumor from pcDNA-tum-5 or pcDNA-tum-5-NGR group was significantly lower than that from pcDNA 3.1 group, with the inhibition rate of 29.4% and 54.1% separately. HE and CD31 staining showed that the anti-angiogenic effect of pcDNA-tum-5-NGR was superior to that of pcDNA-tum-5, suggesting NGR peptide enhanced the Tum-5 activity.Pichia pastoris system is a recently developing eukaryotic system with secretory protein expression, which can simplify the process of purification. Thus, we subclone the genes of Tum-5 and Tum-5-NGR into the vector pPICZaA. After Electroporation and methanol induction, recombinant proteins were detected by SDS-PAGE and western blot.In brief, we firstly constructed recombinant plasmids of pcDNA-tum-5 and pcDNA-tum-5-NGR, which could lead to secretory protein expression in vitro and in vivo. Coculture experiments proved the secreted protein could specifically inhibit the proliferation and migration of endothenial cells. Animal study showed that NGR peptide significantly enhanced the antitumor effect of Tum-5. Furthermore, we evaluated the possibility of secretory expression of such recombinant proteins using Pichia pastoris system, for the fermentation and purification in semi-works production.
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