Recombinant DNA procedures for producing small antimicrobial cationic peptides in bacteria

Kevin L. Piers, Melissa H. Brown, Robert E.W. Hancock

Research output: Contribution to journalArticle

157 Citations (Scopus)

Abstract

Natural polycationic antibiotic peptides have been found in many different species of animals and insects and shown to have broad antimicrobial activity. To permit further studies on these peptides, bacterial expression systems were developed. Attempts to produce these peptides with an N-terminal signal sequence were unsuccessful due to the lability of the basic peptides. Therefore, a number of different fusion protein systems were tested, including fusions to glutathioneS-transferase (GST) (on plasmid pGEX-KP), Pseudomonas aeruginosa outer membrane protein OprF (on plasmid pRW5), Staphylococcus aureus protein A (on plasmid pRIT5), and the duplicated IgG-binding domains of protein A (on plasmid pEZZ18). In the first three cases, stable fusion proteins with the defensin, human neutrophil peptide 1 (HNP-1), and/or a synthetic cecropin/melittin hybrid (CEME) were obtained. In the course of these studies, we developed a novel method of purifying inclusion bodies, using the detergent octyl-polyoxyethylene (octyl-POE), as well as establishing methods for preventing fusion protein proteolytic breakdown. Cationic peptides could be successfully released from the carrier protein with high efficiency by chemical means (CNBr cleavage) and with low efficiency by enzymatic cleavage (using factor Xa protease). Fusions of protein A to cationic peptides were secreted into the culture supernatant of S. aureus clones and after affinity purification, CNBr digestion and column chromatography, pure cationic peptide was obtained. CEME produced by this procedure had the same amino acid (aa) content, aa sequence, gel electrophoretic mobility and antibacterial activity as CEME produced by protein chemical procedures.

Original languageEnglish
Pages (from-to)7-13
Number of pages7
JournalGene
Volume134
Issue number1
DOIs
Publication statusPublished - 30 Nov 1993
Externally publishedYes

Keywords

  • cecropin
  • Defensin
  • expression
  • fusion protein
  • inclusion bodies

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