High efficiency preparation of bioactive human α-defensin 6 in Escherichia coli Origami(DE3)pLysS by soluble fusion expression

Human α-defensin 6 (HD₆), a small cysteine-rich cationic peptide specially expressed in epithelial cells of digestive tract, may play a crucial role in mucosal immunity. This is the first report on efficient production of bioactive HD₆ through a gene-engineering approach in Escherichia coli. The recombinant plasmid pET32a-omHD₆ was primarily constructed by inserting a PCR fragment encoding mature HD₆ peptide (mHD₆) preceded by an enterokinase recognition sequence into the expression vector pET32a(+), in frame with the upstream thioredoxin (TrxA) gene. Under optimized expression conditions, a high percentage (>60%) of soluble TrxA-omHD₆ fusion protein was obtained with a yield of about 1.69 g/l, and the theoretical productivity of recombinant mHD₆ (rmHD₆) reached 0.38 g/l. A feasible three-step purification strategy involving nickel-sepharose chromatography, enterokinase-cleavage and cation exchange chromatography was developed to purify rmHD₆, followed by characteristic identifications by Western blot, mass spectrometry and sequencing. About 102 mg/l of rmHD₆ with its intact N-terminal amino acid sequence was finally achieved. The in vitro experiments showed that rmHD₆ possesses high potency to inhibit herpes simplex virus-2 infection. This work settles substantial foundation for further functional study of HD₆.