title: High efficiency preparation of bioactive human alpha-defensin 6 in Escherichia coli Origami(DE3)pLysS by soluble fusion expression.
abstract: Human alpha-defensin 6 (HD(6)), 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 HD(6) through a gene-engineering approach in Escherichia coli. The recombinant plasmid pET32a-omHD(6) was primarily constructed by inserting a PCR fragment encoding mature HD(6) 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 HD(6).
authors.affiliation: State Key Laboratory of Trauma, Burns and Combined Injury, Institute of Combined Injury of PLA, Third Military Medical University, Chongqing 400038, People's Republic of China.
authors.forename: Aiping
authors.initials: A
authors.lastname: Wang
authors.forename: Yongping
authors.initials: Y
authors.lastname: Su
authors.forename: Song
authors.initials: S
authors.lastname: Wang
authors.forename: Mingqiang
authors.initials: M
authors.lastname: Shen
authors.forename: Fang
authors.initials: F
authors.lastname: Chen
authors.forename: Mo
authors.initials: M
authors.lastname: Chen
authors.forename: Xinze
authors.initials: X
authors.lastname: Ran
authors.forename: Tianmin
authors.initials: T
authors.lastname: Cheng
authors.forename: Junping
authors.initials: J
authors.lastname: Wang
mesh_terms: D015153:Blotting, Western; D002460:Cell Line; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004765:Enteropeptidase / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D015870:Gene Expression; D005822:Genetic Vectors; D018258:Herpesvirus 2, Human / Q000187:drug effects; D006801:Humans; D013058:Mass Spectrometry; D008826:Microbial Sensitivity Tests; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000494:pharmacology; D011995:Recombination, Genetic; D017421:Sequence Analysis; D023084:alpha-Defensins / Q000302:isolation & purification* / Q000494:pharmacology
chemical_list: C517765:DEFA6 protein, human; D011993:Recombinant Fusion Proteins; D023084:alpha-Defensins; D004765:Enteropeptidase