DETERMINANTS OF RECOMBINANT PRODUCTION OF ANTIMICROBIAL CATIONIC PEPTIDES AND CREATION OF PEPTIDE VARIANTS IN BACTERIA

Cationic peptides possessing antibacterial activity are virtually ubiq uitous in nature, and offer exciting prospects as new therapeutic agen ts. We had previously demonstrated that such peptides could be produce d by fusion protein technology in bacteria and several carrier protein s had been tested as fusion partners including glutathione-S-transfera se, S. aureus protein A, IgG binding protein and P. aeruginosa outer m embrane protein OprF. However these fusion partners, while successfull y employed in peptide expression, were not optimized for high level pr oduction 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 sma ll replication protein RepA from E. coli and used its truncated versio n to construct fusion partners. The minimal elements required for high level expression of cationic peptide were defined as a DNA sequence e ncoding a fusion protein comprising, from the N-terminus, a 68 amino a cid 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 cell ulose binding domain and a hexa histidine domain. The improved system showed high level expression and simplified downstream purification. T he active peptide could be yielded by CNBr cleavage of the fusion prot ein. This novel vector was used to express three classes of cationic p eptides including the alpha-helical peptide CEMA, the looped peptide b actenecin and the extended peptide indolicidin. In addition, mutagenes is of the peptide gene to produce peptide variants of CEMA and indolic idin using the improved vector system was shown to be successful. (C) 1998 Academic Press.