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One prominent class of cationic antibacterial peptides comprises the alpha-helical class, which is unstructured in free solution but folds into an amphipathic alpha-helix upon insertion into the membranes of target cells. To investigate the importance of alpha-helicity and its induction on interaction with membranes, a series of peptides was constructed based on a hybrid of moth cecropin (amino acids 1-8) and bee melittin (amino acids 1-18) peptides. The new peptides were predicted to have a high tendency to form alpha-helices or to have preformed alpha-helices by virtue of construction of a lactam bridge between glutamate and lysine side-chains at positions i and i + 4 at various locations along the primary sequence. In two examples where the use of lactam bridge constraints induced and stabilized alpha-helical structure in benign (aqueous buffer) and/or hydrophobic medium, there was a decrease in antibacterial activity relative to the linear counterparts. Thus the preformation of alpha-helix in solution was not necessarily beneficial to antimicrobial activity. In the one case where the lactam bridge did result in increased antibacterial activity (lower minimal inhibitory concentration values) it did not increase alpha-helical content in benign or hydrophobic medium. Broadly speaking, good activity of the peptides against Pseudomonas aeruginosa correlated best (r2 = 0.88) with a helican parameter which was calculated as the induction of alpha-helix in a membrane-mimicking environment divided by the alpha-helix formation under benign conditions. Interestingly, the activity of the lactam bridge peptide constructs correlated in part with alterations in bacterial outer or cytoplasmic membrane permeability.

Cationic peptides possessing antibacterial activity are virtually ubiquitous in nature, and offer exciting prospects as new therapeutic agents. We had previously demonstrated that such peptides could be produced by fusion protein technology in bacteria and several carrier proteins had been tested as fusion partners including glutathione-S-transferase, S. aureus protein A, IgG binding protein and P. aeruginosa outer membrane protein OprF. However these fusion partners, while successfully employed in peptide expression, were not optimized for high level production of cationic peptides (Piers, K., Brow, M. L., and Hancock, R. E. W. 1993, Gene 137, 7-13). In this paper we took advantage of a small replication protein RepA from E. coli and used its truncated version to construct fusion partners. The minimal elements required for high level expression of cationic peptide were defined as a DNA sequence encoding a fusion protein comprising, from the N-terminus, a 68 amino acid carrier region, an anionic prepro domain, a single methionine and the peptide of interest. The 68 amino acid carrier region was a block of three polypeptides consisting of a truncated RepA, a synthetic cellulose binding domain and a hexa histidine domain. The improved system showed high level expression and simplified downstream purification. The active peptide could be yielded by CNBr cleavage of the fusion protein. This novel vector was used to express three classes of cationic peptides including the alpha-helical peptide CEMA, the looped peptide bactenecin and the extended peptide indolicidin. In addition, mutagenesis of the peptide gene to produce peptide variants of CEMA and indolicidin using the improved vector system was shown to be successful.