Improving Soluble Expression of Curcin-Transferrin Receptor-binding Peptide Fusion Protein with Glutathione Transferase-Small Ubiquitin-related Modifier System

Abstract

Curcin can inhibit the proliferation of tumor cells and facilitate tumor cell apoptosis. In the current study, transferrin receptor (TFR)-binding peptide, TfRBP9, was fused with curcin and significantly enhanced the targeting of the anti-tumor ability of curcin. However, the recombinant curcin-TfRBP9 was expressed as an inclusion body in bacteria. The active recombinant curcin-TfRBP9 was obtained through dissolution, purification and renaturation. To achieve the soluble expression of recombinant curcin-TfRBP9, the vector pQE-30 inserted into a deoxyribonucleic acid (DNA) coding segment with a glutathione transferase (GST) -small ubiquitin-related modifier (SUMO)-curcin-TfRBP9 fusion protein was transferred into Escherichia coli (E. coli ) M15. After being induced by 0.5 mM of isopropyl-b-D-thiogalactoside (IPTG) for 20 h at 20°C, the expressed quantity of the GST-SUMO-curcin-TfRBP9 fusion protein was about 40% of the total protein, and the soluble expression of the recombinant protein was about 70% of the total GST-SUMO-curcin-TfRBP9 fusion protein. Subsequent purification through Glutathione Sepharose 4B, ubiquitin-like specific protease1 (Ulp1) digestion and CM SepharoseTM Fast Flow yielded the untagged curcin-TfRBP9 fusion protein with a purity of more than 95%. The curcin-TfRBP9 fusion protein had significant proliferation inhibitory effects on the lung cancer A549 cells that over-expressed transferrin receptors, and it had lower inhibitory effects on normal human LO-2 liver cells. Compared with the control untagged curcin, the curcin-TfRBP9 protein promoted higher inhibition rates in the A549 cells.

College of Pharmacy,Core tip: A method to facilitate soluble expression of curcin-TfRBP9 fusion protein was achieved by application of GST and SUMO tags. Subsequent purification through Glutathione Sepharose 4B, ubiquitin-like specific protease1 (Ulp1) digestion and CM SepharoseTM Fast Flow yielded the untagged curcin-TfRBP9 fusion protein with a purity of more than 95%. The MTT assay showed that untagged curcin-TfRBP9 fusion had significant proliferation inhibitory effects on the lung cancer A549 cells that over-expressed transferrin receptors, and it had lower inhibitory effects on normal human LO-2 liver cells. Compared with the control untagged curcin, the curcin-TfRBP9 protein promoted higher inhibition rates in the A549 cells.