Expression and purification of recombinant human alpha-defensins in Escherichia coli.
Different strategies have been developed to produce small antimicrobial peptides (AMPs) using recombinant techniques. Up to now, all efforts to obtain larger quantities of active recombinant human alpha-defensins have been only moderately successful. Here we report an effective method of biosynthesis of human alpha-defensins (hNP-1 to hNP-3 and hD-5 and hD-6) in the Escherichia coli. All the peptides, expressed as insoluble fusions with the peptide encoded by a portion of E. coli tryptophan operon (trp DeltaLE 1413 polypeptide), were isolated from the inclusion bodies by immobilized metal affinity chromatography (IMAC) and separated from the fusion leader by chemical cleavage. Fully reduced peptides that were purified according to a straightforward protocol were subsequently folded, oxidized, and subjected to functional and structural analyses. With the exception of hD-6, all recombinant alpha-defensins exhibit expected anti-E. coli activity, as measured by the colony counting method. The method described in this report is a low-cost, efficient way of generating alpha-defensins in quantities ranging from milligrams to grams.
Pazgier
Marzena
M
Macromolecular Crystallography Laboratory, National Cancer Institute at Frederick, Frederick, MD 21702, USA.
Lubkowski
Jacek
J
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D001483:Base Sequence; D017124:Conserved Sequence; D004926:Escherichia coli / Q000187:drug effects / Q000378:metabolism*; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D016415:Sequence Alignment; D023084:alpha-Defensins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification* / Q000494:pharmacology
D000900:Anti-Bacterial Agents; D010446:Peptide Fragments; D011994:Recombinant Proteins; D023084:alpha-Defensins