Antibiotic peptide cecropin B2

In this study, genetic engineering was applied to the overexpression of the antimicrobial peptide (AMP) cecropin B2 (cecB2). pTWIN1 vector with a chitin-binding domain (CBD) and an auto-cleavage Ssp DnaB intein (INT) was coupled to the cecB2 to form a fusion protein construct and expressed via Escherichia coli ER2566. The cecB2 was obtained via the INT cleavage reaction, which was highly related to its adjacent amino acids. Three oligopeptide cleavage variants (OCVs), i.e., GRA, CRA, and SRA, were used as the inserts located at the C-terminus of the INT to facilitate the cleavage reaction. SRA showed the most efficient performance in accelerating the INT self-cleavage reaction. In addition, in order to treat the INT as a biocatalyst, a first-order rate equation was applied to fit the INT cleavage reaction. A possible inference was proposed for the INT cleavage promotion with varied OCVs using a molecular dynamics (MD) simulation. The production and purification via the CBD-INT-SRA-cecB2 fusion protein resulted in a cecB2 yield of 58.7 mg/L with antimicrobial activity.

Cecropin B2 (CecB) is a 35 amino acid residue, antibacterial peptide that was isolated from the hemolymph and cuticular matrix of the silkworm, Bombyx mori. Synthetic peptides with sequences corresponding to CecB and its truncated analogue, [3-->35]CecB, were synthesized and selectively labeled at their N-terminal amino acids with either 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) or rhodamine fluorescent probes. Utilization of these probes facilitated study of the interaction of cecropin with model phospholipid membranes at a high lipid/peptide molar ratio (approximately 3000:1), permitting investigation of the initial steps involved in this process. The surface partition coefficient of CecB, derived from binding isotherms of the NBD-labeled peptide, was 10-fold higher with acidic phospholipids than with zwitterionic ones, which correlates with the high efficiency of CecB and its analogues in permeating acidic phospholipid vesicles. Furthermore, a direct correlation was found between the antibacterial activity of CecB or its truncated analogues and the ability of their Rho-labeled analogues to interact with bacteria and human red blood cells. We propose that CecB binds phospholipid membranes preferentially as monomers lying on the surface, rather than cooperatively as bundles that form transmembranal pores via a "barrel stave" mechanism. This is based on the following: (i) the linearity of CecB's binding isotherms; (ii) the low energy transfer between membrane-embedded donor and acceptor-labeled CecB, even in the presence of a transmembrane potential; (iii) the surface localization of CecB's N-terminus; (iv) the need for more than 100 peptide molecules per phospholipid vesicle to induce initial ion leakage; and (v) the fact that CecB is a highly positively charged amphipathic alpha-helix, and therefore it is not expected to transverse the membrane as a monomer. We speculate that the non-cooperative binding of the peptides on the outer surface of the bacteria (i.e., no aggregation of CecB monomers) may help them to diffuse efficiently into the inner membrane, which is thought to be the target of antibacterial peptides.

Custom designed analogs of the natural anti-bacterial peptide cecropin B (CB) have been synthesized; cecropin B-1 (CB-1) was constructed by replacing the C-terminal segment (residues 26 to 35) with the N-terminal sequence of CB (positions 1 to 10 which include five lysine residues). The second analog, CB-2, is identical to CB-1 except for the insertion of a Gly-Pro residue pair between Pro-24 and Ala-25. These peptides were used to investigate their anti-liposome, anti-bacterial and anti-cancer activities. The strength of anti-liposome activity is reduced two- to three-fold when the analogs are used instead of natural CB based on DL50 analysis. Similarly, the potency of these analogs on certain bacteria is about two- to four-fold lower than those of CB based on LC measurements. In contrast, on leukemia cancer cells, the potency of CB-1 and CB-2 is about two- to three-fold greater than that of natural CB based on IC50 measurements. All CB, CB-1 and CB-2 peptides have comparable helix contents according to CD measurements. These results indicate that the designed cationic lytic peptides, having extra cationic residues, are less effective in breaking liposomes and killing bacteria but more effective in lysing cancer cells. The possible interpretations for these observations are discussed.