Expression of Kininogen D560-148-TRAIL114-281 fusion protein and its angiogenesis-inhibiting and apoptosis-inducing effect

Objective: To express Kininogen D560-148-TRAIL114-281 fusion protein using prokaryotic system and observe its biological functions. Methods: The Kininogen D560-148 gene and TNF-related apoptosis-inducing ligand (TRAIL114-281) gene were amplified by PCR and were cloned into pMAL expression vector to construct recombinant pMAL-Kininogen DS60-148 (PMAL-KD5), pMAL-TRAIL114-281 (pMAL-TRAIL) and pMAL-Kininogen D560-148-TRAIL114-281 (pMAL-KT) plasmids, respectively. The plasmids were transformed into E. coli BL21 and were efficiently expressed after IPTG induction. The purified MBP-KD5, MBP-TRAIL and MBP-KT proteins were obtained by amylose resin affinity purification column. The proliferation of cells was measured by MTT; tube formation of endothelial cell was detected by tube formation assay; and the apoptosis of cells were observed by electron microscopic and FCM. Results: Prokaryotic expression vectors pMAL-KD5, pMAL-TRAIL and pMAL-KT and their purified fusion proteins MBP-KD5, MBP-TRAIL and MBP-KT were successfully obtained. MBP-KT significantly inhibited the proliferation of ECV304 endothelial cells, SW1990 pancreatic cancer cells and the tube formation of ECV304 cells compared with those of MBP-KD5 and MBP-TRAIL. Meanwhile, MBP-KT dose-dependently induced the apoptosis of SW1990 cells. Conclusion: Kininogen D5 60-148-TRAIL114-281 fusion protein can inhibit the proliferation of tumor cells and angiogenesis of endothelial cells, which lays a foundation for further research on tumor-targeting drugs.