RECOMBINANT-DNA PROCEDURES FOR PRODUCING SMALL ANTIMICROBIAL CATIONICPEPTIDES IN BACTERIA

Natural polycationic antibiotic peptides have been found in many diffe rent species of animals and insects and shown to have broad antimicrob ial activity. To permit further studies on these peptides' bacterial e xpression systems were developed. Attempts to produce these peptides w ith an N-terminal signal sequence were unsuccessful due to the labilit y of the basic peptides. Therefore, a number of different fusion prote in systems were tested, including fusions to glutathione-S-transferase (GST) (on plasmid pGEX-KP), Pseudomonas aeruginosa outer membrane pro tein OprF (on plasmid pRW5), Staphylococcus aureus protein A (on plasm id pRIT5), and the duplicated IgG-binding domains of protein A (on pla smid pEZZ18). In the first three cases, stable fusion proteins with th e defensin, human neutrophil peptide 1 (HNP-1), and/or a synthetic cec ropin/melittin hybrid (CEME) were obtained. In the course of these stu dies, we developed a novel method of purifying inclusion bodies, using the detergent octyl-polyoxyethylene (octyl-POE), as well as establish ing methods for preventing fusion protein proteolytic breakdown. Catio nic peptides could be successfully released from the carrier protein w ith high efficiency by chemical means (CNBr cleavage) and with low eff iciency by enzymatic cleavage (using factor X(a) protease). Fusions of protein A to cationic peptides were secreted into the culture superna tant of S. aureus clones and after affinity purification, CNBr digesti on and column chromatography, pure cationic peptide was obtained. CEME produced by this procedure had the same amino acid (aa) content, aa s equence, gel electrophoretic mobility and antibacterial activity as CE ME produced by protein chemical procedures.