Expression and purification of human antimicrobial peptide, dermcidin, in Escherichia coli.
Human dermcidin, an anionic antimicrobial peptide expressed in the pons of the brain and the sweat glands, displays antimicrobial activity against pathogenic microorganisms such as Staphylococcus aureus and Candida albicans. Here, we describe the recombinant production of a 48 amino acid dermcidin variant with C-terminal homoserine lactone (DCD-1Hsl). Dermcidin coding sequence was cloned downstream of a 125 amino acid ketosteroid isomerase gene and upstream of a His6Tag sequence in pET-31b(+) vector and transformed into Escherichia coli. The fusion protein was expressed in the form of inclusion bodies, purified on His Bind Resin, and cleaved by CNBr to release recombinant DCD-1Hsl. Purification of rDCD-1Hsl was achieved by solid-phase extraction that yielded milligram amounts of peptide with more than 95% purity. Recombinant peptide showed antimicrobial activities against E. coli ML-35p, Salmonella typhimurium 5156, Listeria monocytogenes 264, S. aureus 29/58 (clinical isolate), and C. albicans K2 (clinical strain). The application of this expression/purification approach represents a fast and efficient method to prepare milligram quantities of dermcidin in its biologically active form.
Cipáková
Ingrid
I
CPN spol. s r.o., 561 02 Dolní Dobrouc 401, Czech Republic. icipakova@yahoo.com
Gasperík
Juraj
J
Hostinová
Eva
E
D000595:Amino Acid Sequence; D000890:Anti-Infective Agents / Q000302:isolation & purification / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry / Q000378:metabolism; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D010455:Peptides / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D000890:Anti-Infective Agents; D010455:Peptides; D011993:Recombinant Fusion Proteins; C442243:dermcidin