Productive folding of human neutrophil alpha-defensins in vitro without the pro-peptide.
Human neutrophil alpha-defensins (HNPs) are small, Cys-rich, cationic antimicrobial proteins. Stored in the azurophilic granules of neutrophils, they are released during phagocytosis to kill ingested foreign microbes through disruption of their cytoplasmic membrane. Recently, the three most abundant forms of human alpha-defensins, HNPs 1-3, have been implicated in suppressing HIV-1 infection in vivo, thereby exhibiting a potential therapeutic value in the treatment of AIDS. HNPs are synthesized as inactive precursors in vivo and require proteolytic removal of their inhibitory N-terminal pro-peptide for activation. Folding of HNPs 1-3 in vitro without the pro-peptide has been reported to be extremely difficult, which led to the hypothesis that the 45-residue anionic pro-peptide may assist proHNPs folding as an intramolecular chaperone interacting with the cationic C-terminal domain, a mechanism reminiscent of some bacterial serine proteases. Here we show that HNPs without the pro-region can fold productively with yields over 80% in the presence of 2 M urea and 25% N,N-dimethylformamide (DMF). Our finding demonstrates an efficient protocol for the production of large quantities of highly pure human alpha-defensins and is broadly applicable in folding aggregation-prone, Cys-rich proteins of both synthetic and recombinant origin.
Wu
Zhibin
Z
Institute of Human Virology, University of Maryland Biotechnology Institute, 725 West Lombard Street, Baltimore, MD 21201, USA.
Powell
Robert
R
Lu
Wuyuan
W
D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000494:pharmacology; D003545:Cysteine / Q000737:chemistry; D004926:Escherichia coli / Q000187:drug effects; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008826:Microbial Sensitivity Tests; D017510:Protein Folding; D011498:Protein Precursors / Q000737:chemistry*; D017433:Protein Structure, Secondary; D013302:Streptomyces / Q000187:drug effects; D023084:alpha-Defensins / Q000737:chemistry*
D000900:Anti-Bacterial Agents; D011498:Protein Precursors; D023084:alpha-Defensins; C406413:human neutrophil peptide 1; D003545:Cysteine