Journal
APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
Volume 87, Issue 5, Pages 1935-1942Publisher
SPRINGER
DOI: 10.1007/s00253-010-2688-y
Keywords
Human alpha-defensin 6; Expression; Purification; Escherichia coli; Antiviral activity
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Funding
- National Natural Science Foundation of China [30771892]
- Academician Fund of Chongqing City [2007AB5022]
- Special Fund of National Key Laboratory of Trauma, Burn and Combined Injury [SKLZZ200821]
- National 863 High-tech Development Plan of China [2007AA02Z152]
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Human alpha-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-omHD(6) was primarily constructed by inserting a PCR fragment encoding mature HD6 peptide (mHD(6)) 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-omHD(6) fusion protein was obtained with a yield of about 1.69 g/l, and the theoretical productivity of recombinant mHD(6) (rmHD(6)) 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 rmHD(6), followed by characteristic identifications by Western blot, mass spectrometry and sequencing. About 102 mg/l of rmHD(6) with its intact N-terminal amino acid sequence was finally achieved. The in vitro experiments showed that rmHD(6) possesses high potency to inhibit herpes simplex virus-2 infection. This work settles substantial foundation for further functional study of HD6.
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