论文部分内容阅读
Trichosanthin (TCS), originally extracted from a herbs of traditional Chinese medicine, is a type Ⅰ ribosome-inactivating protein.It has been well-established that TCS has evident anti-tumor effects largely by inducing apoptosis of cancer cells and currently TCS is an approved clinical drug in China.However, the clinical application of native TCS is limited by its poor tumor-targeting and cell-penetrating due to the various distribution of its specific cellular receptors.To address this issue, a genetically engineered TCS was constructed in which TCS was fused with a recently invented supercharged green fluorescent protein (scGFP, provided by Dr.David Liu of Harvard University) based on the new discovery that scGFP-tagged fusion protein acquired ability to penetrate cells.The fusion gene of TCS-scGFP was inserted into the prokaryotic expression vector pET (Novagen) and the recombinant plasmid was transformed into E.coli BL21 (DE3)pLysS, then the fusion protein expression was induce by IPTG and subsequently purified by Ni-NTA system following Qiagens protocol.It turned out that a novel fusion protein TCS-scGFP was prokaryotically expressed and purified successfully, as demonstrated by Western blot.In addition, the experiments of Coomassie brilliant blue staining and density scanning showed that the expressed protein accounted for >50% of total proteins.The pilot experiment indicated that the TCS-scGFP fusion protein was able to transduce HeLa cancer cells efficiently.It is expected the novel TCS-scGFP fusion protein will overcome the limitations of native TCS and the further studies are in progress.