Biotechnology Bulletin ›› 2018, Vol. 34 ›› Issue (6): 204-212.doi: 10.13560/j.cnki.biotech.bull.1985.2017-1119

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Screening of a Gelonin Fusion Protein with High Cell-penetrating Efficiency and Its Anti-tumor Activity and Apoptosis Pathway

ZHAI Yi-zhou1 ,LU Mei-ya2 ,ZHAO Jian1 ,WANG Fu-jun2,3   

  1. 1. State Key Laboratory of Bioreactor Engineering,East China University of Science and Technology,Shanghai 200237;
    2. Zhejiang Reachall Pharmaceutical Co. Ltd,Dongyang 322100;
    3. Institute of Chinese Materia Medica,Shanghai University of Traditional Chinese Medicine,Shanghai 201203
  • Received:2017-12-26 Online:2018-06-26 Published:2018-07-03

Abstract: By fusing gelonin with a variety of cell penetrating peptides(CPP),a recombinant protein gelonin-HBP with the highest anti-tumor activity was obtained and its molecular mechanism of inhibiting tumor cell growth was also studied. The recombinant protein of gelonin fused with four different CPPs(R9,TAT,HBD,and HBP)was expressed in Escherichia coli BL21(DE3)and then purified by NI-NTA affinity chromatography. Superhelical cleavage experiment and in vitro translation inhibition assay were used to detect the bioactivity of gelonin fusion protein. The translocation efficiency of gelonin recombinant protein was observed by laser scanning confocal microscopy. The cell growth inhibition effect of gelonin fusion protein on tumor cells was detected by MTT assay and the molecular mechanism of cell apoptosis was observed by flow cytometry and Western blot. The results showed that four gelonin recombinant proteins that retained the original biological activity of gelonin were obtained,and the fusion with a CPP promoted the cell penetration of gelonin,improved the inhibition effect of gelonin on tumor cells,and accelerated the apoptosis of these tumor cells. Compared to gelonin alone,the gelonin-HBP fusion protein increased its potency in 4 tumor cells of HeLa,B16,MCF-7 and HepG2 by 16.4 times,9.6 times,14.0 times and 24.6 times,respectively. Western blotting analysis demonstrated that gelonin-HBP induced an enhancement in the activity of Caspas 3,Caspas 8 and Caspas 9 in tumor cells,thus the expression of Bax significantly increased,while the expression of Bcl-2 significantly decreased. Conclusively,CPP may enhance the anti-tumor effect and the apoptosis of gelonin on tumor cells;among them,HBP,a CPP derived from human heparin-binding epidermal growth factor-like growth factor,makes the gelonin-HBP have the strongest anti-tumor effect. The mechanism of inducing tumor cell apoptosis by this recombinant protein is related to mitochondrial and death receptor-mediated apoptosis signaling pathway.

Key words: gelonin, CPPs, anti-tumor, cell apoptosis