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Expression of Buckwheat Protease Inhibitor (aBTI) and Its Antitumor Efficacy against Human Hepatoma (HepG2) Cells
Xin Tian, Yu-Ying Li, Zhuan-Hua Wang*
Key Laboratory for Chemical Biology and Molecular Engineering of Ministry of Education, Institute of Biotechnology, Shanxi University, Taiyuan 030006, China
Abstract: Previous studies have shown that recombinant buckwheat trypsin inhibitor (rBTI) possesses potent antitumor activity in in vitro models. In this study， a mutant (R45A-aBTI) of rBTI was generated through site-directed mutagenesis. aBTI was expressed in a soluble state in E.coli. M15 [pREP4] and purified by affinity chromatography and gel filtration chromatography. The purity of the aBTI reached above 95%. Activity analysisindicated that aBTI had a specific inhibitory activity toward elastase， its inhibition constant (Ki) value was 3.34 × 10-7 mol/L. MTT assay demonstrated that rBTI could inhibit the growth of HepG2 tumor cells in a dose-dependent manner (IC50:1.88 μmol/L). Additionally， nuclear staining showed that aBTI could induce the apoptosis of HepG2 cells. Our results suggested that aBTI displayed more marked antitumor efficacy in in vitro and might be a novel candidate for cancer treatment.
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