Expression Of RBTI-2 And Its Anti-tumor Activity

Trypsin inhibitor (TI) is widely present in plants, animals and microorganisms. TI play a critical role in lots of physiology systems, and they are significant element in maintain a stability internal environment. So far, a variety of TI has been isolated and characterized gradually, and their structure and function have also been conducted in-depth study. A number of TIs are widely applied in fields of agriculture and medicine. Especially, as a potential anticancer drug, increasing number researchers have focused their aims on trypsin inhibitor.Currently protease inhibitor drugs already developed mostly from people and animals, such as human urinary trypsin inhibitor (UTI), aprotinin, however, due to their molecular weight, complex structure, separation and extraction are difficult and so on, there are many inadequacies in terms of treatment. Plant sources of TIs have been widely applied, which have small molecular weight, rich sources and versatile features. According to the amino acid sequence homology, trypsin inhibitors can be divided into Bowman-Birk, Kunitz, PotatoⅠ, PotatoⅡ, Serpin, Cereal STI and other families, most studies of inhibitors are focus on Bowman-Birk, Kunitz and PotatoⅠfamilies at present.Buckwheat is a traditional farm crops of our country, and its contains a rich supply of amino acids, which could reduce cholesterin, prevent cardiopathy and cancer, resist caducity, reduce blood pressure and improving diabetic etc, so as a kind of health food, buckwheat has been received more and more people's favor. Previously, we had isolated and purified buckwheat trypsin inhibitor (BTI) from buckwheat seeds, and amplified the buckwheat trypsin inhibitor gene through RT-PCR and RACE methods. We had expressed and purified a recombinant buckwheat trypsin inhibitor (rBTI). In vitro, rBTI can inhibit the proliferation to several types of human tumor cells, while it has shows less toxicity against normal human cells, and rBTI can induce the apoptosis through the mitochondrial apoptotic pathway. Thus as a potential anticancer drugs, further investigation is necessary to fully characterize the buckwheat trypsin inhibitor.To design a recombinant buckwheat trypsin inhibitor(rBTI-2) of without label that suitable for developing genetic engineering, in this study, we selecte the expression vector pExSecI, and constructe the pExSecI-BTI-2. Soluble protein could acquire through inducing by IPTG. After purified through ResourceTM Q column, the possible effects of rBTI-2 on the proliferation of tumor cells were investigated by MTT assays. The results indicated that rBTI-2 could specifically inhibit the growth of HepG2, EC9706 and QBC-939 in a dose-dependent manner, while it had showed less toxicity against HL-7702 cells. The wound-healing assay showed that the low concentration rBTI-2 could suppress HepG2 cells invasion on different level. In addition, we compared the effect of rBTI-2 and several fusion proteins on tumor cells. The results indicated that rBTI-2, rBTI, aBTI, fBTI and R,D-rBTI could inhibit the proliferation of tumor cells on different level a dose-dependent manner also. Comparing with fusion protease inhibitors (rBTI), the inhibit proliferation activity on tumor cells of rBTI-2 was slightly strong. In order to mass production of rBTI-2 proteins, we optimize its expression conditions. In conclusion, the suitble time and the concentration of IPTG were 3.5 h and 0.5 mmol/L, respectively. At the same time, we could obtain high yields of rBTI-2 proteins.All above results can provid important foundation and evidence for the molecule mechanism of apoptosis and application of rBTI-2.