Abstract: Thanatin is one of the antibacterial peptides which consists of 21 amino acids and has a broad spectrum of antimicrobial activities. To explore a cost-effective approach for highlevel expression of the thanatin in Escherichia coli, the cDNA fragment encoding thanatin with preferred codons of E. coli was obtained by recursive PCR (rPCR) and fused to the C-terminal of ubiquitin (UBI) from housefly, Musca domestica. The fused gene was inserted into the plasmid pET32a to construct the expression vector pET-TRX-UBI-THA and express the fusion protein with 6×His tag in E. coli BL21. The fusion protein was expressed in soluble form under the optimized conditions at a high-level (more than 46% of the total proteins). With 6×His tag, the proteins were easily purified by Ni²⁺-NTA chromatography. The purified proteins were efficiently cleaved by ubiquitin carboxyl-terminal hydrolase which linked with 6×His tag at the carboxyl-terminus (UCH-6×His), yielding recombinant thanatin with significant antimicrobial activity. After contaminants removal by Ni²⁺-NTA chromatography, recombinant thanatin was purified by reversed phase HPLC, 5.4 mg of pure active thanatin was obtained from 1 L culture medium. ESI-MS analysis showed that the molecular weight of the purified recombinant thanatin was 2.57 kD, which perfectly matched the mass calculated from the amino acid sequence. The antimicrobial assay showed that the purified thanatin has high activities against E. coli K12D31 and Staphylococcus aureus. Our results demonstrated that functional thanatin can be produced in sufficient quantity using the ubiquitin fusion technique at a low cost.

Key words: Antibacterial peptides, thanatin, ubiqutin, fusion expression, purification, bioassay