Hinnavin I

Hinnavins, together with lysozymes, are the main types of antibacterial peptides/proteins previously isolated from the larval haemolymph of the cabbage butterfly, Artogeia rapae as part of the humoral immune response to a bacterial invasion. One of these antibacterial peptides, named hinnavin II, was purified and characterized after cDNA cloning. The purified hinnavin II was more active against Gram negative than against Gram positive bacteria. Hinnavin II also showed a powerful synergistic effect on the inhibition of bacterial growth with purified lysozyme. The cDNA has a total length of 186 bp with a 114 coding region. The deduced protein sequence contains 38 amino acids with a coding capacity of 4142.8 Da. The result of a multiple sequence alignment and phylogenetic analysis with Clustal W indicated that mature hinnavin II showed an approximately 78.9% amino acid sequence identity with cecropin A and originated from a group containing mostly lepidopteran cecropins.

The increasing problem of antibiotic resistance among pathogenic bacteria requires novel strategies for the construction of multiple, joined genes of antimicrobial agents. The strategy used in this study involved synthesis of a cDNA-encoding hinnavin II/alpha-melanocyte-stimulating hormone (hin/MSH) hybrid peptide, which was cloned into the pET32a (+) vector to allow expression of the hybrid peptide as a fusion protein in Escherichia coli BL21 (DE3). The resulting expression of fusion protein Trx-hin/MSH could reach up to 20% of the total cell proteins. More than 50% of the target protein was in a soluble form. The target fusion protein from the soluble fraction, Trx-hin/MSH, was easily purified by Ni(2+)-chelating chromatography. Then, enterokinase cleavage effectively cleaved the Trx-hin/MSH to release the recombinant hin/MSH (rhin/MSH) hybrid peptide. After removing the contaminants, we purified the recombinant hybrid peptide to homogeneity by reversed-phase FPLC and obtained 210 mg of pure, active rhin/MSH from 800 ml of culture medium. Antimicrobial activity assay demonstrated that rhin/MSH had a broader spectrum of activity than did the parental hinnavin II or MSH against fungi and Gram-positive and Gram-negative bacteria. These results suggest an efficient method for producing high-level expression of various kinds of antimicrobial peptides that are toxic to the host, a reliable and simple method for producing different hybrid peptides for biological studies.

We have previously isolated four antibacterial peptides from the immune haemolymph of the fifth instar larvae of cabbage butterfly, Artogeia rapae [Yoe, S. M., Bang, I. S., Kang, C. S., and Kim, H. J. (1996) Mol. Cells 6, 609-614]. They were induced by live, nonpathogenic gram negative bacteria. One of these novel antibacterial peptides was named hinnavin I. Hinnavin I is heat stable; its activity was retained after 60 min incubation at 100 degrees C, being effective against gram negative and/or gram positive bacteria. Hinnavin I and lysozyme II showed a powerful synergistic effect on the inhibition of bacterial growth. Amino acid composition was analyzed and the molecular weight was determined to be 4,139.94+/-10.91 Da by the ESI mass spectrometer. To elucidate the primary structure of hinnavin I, the amino acid sequence of this peptide was determined by N-terminal sequencing techniques. The amino-terminal half of the molecule was rich in charged amino acids and was hydrophilic, whereas the carboxyl-terminal half was hydrophobic.