Mitsuhiro Miyazawa

In Vitro Antimicrobial Properties of Recombinant ASABF, an Antimicrobial Peptide Isolated from the Nematode Ascaris suum

ABSTRACT ASABF is a CSαβ-type antimicrobial peptide that contains four intramolecular disulfide bridges (Y. Kato and S. Komatsu, J. Biol. Chem. 271:30493–30498, 1996). In the present study, a recombinant ASABF was produced by using a yeast expression system, and its antimicrobial activity was characterized in detail. The recombinant ASABF was active against all gram-positive bacteria tested (7 of 7; minimum bactericidal concentration [MBC], 0.03 to 1 μg/ml) exceptLeuconostoc mesenteroides, some gram-negative bacteria (8 of 14; MBC, >0.5 μg/ml), and some yeasts (3 of 9; MBC >3 μg/ml). Slight hemolytic activity (4.2% at 100 μg/ml) against human erythrocytes was observed only under low-ionic-strength conditions. Less than 1 min of contact was enough to kill Staphylococcus aureus ATCC 6538P. The bactericidal activity against S. aureus was inhibited by salts.

Overexpression of an antimicrobial peptide derived from C. elegans using an aggregation-prone protein coexpression system

Antibacterial factor 2 (ABF-2) is a 67-residue antimicrobial peptide derived from the nematode Caenorhabditis elegans. Although it has been reported that ABF-2 exerts in vitro microbicidal activity against a range of bacteria and fungi, the structure of ABF-2 has not yet been solved. To enable structural studies of ABF-2 by NMR spectroscopy, a large amount of isotopically labeled ABF-2 is essential. However, the direct expression of ABF-2 in Escherichia coli is difficult to achieve due to its instability. Therefore, we applied a coexpression method to the production of ABF-2 in order to enhance the inclusion body formation of ABF-2. The inclusion body formation of ABF-2 was vastly enhanced by coexpression of aggregation-prone proteins (partner proteins). By using this method, we succeeded in obtaining milligram quantities of active, correctly folded ABF-2. In addition, 15 N-labeled ABF-2 and a well-dispersed heteronuclear single quantum coherence (HSQC) spectrum were also obtained successfully. Moreover, the effect of the charge of the partner protein on the inclusion body formation of ABF-2 in this method was investigated by using four structurally homologous proteins. We concluded that a partner protein of opposite charge enhanced the formation of an inclusion body of the target peptide efficiently.

Fiber formation of a synthetic spider peptide derived from Nephila clavata

Dragline silk is a high‐performance biopolymer with exceptional mechanical properties. Artificial spider dragline silk is currently prepared by a recombinant technique or chemical synthesis. However, the recombinant process is costly and large‐sized synthetic peptides are needed for fiber formation. In addition, the silk fibers that are produced are much weaker than a fiber derived from a native spider. In this study, a small peptide was chemically synthesized and examined for its ability to participate in fiber formation. A short synthetic peptide derived from Nephila clavata was prepared by a solid‐phase peptide method, based on a prediction using the hydrophobic parameter of each individual amino acid residue. After purification of the spider peptide, fiber formation was examined under several conditions. Fiber formation proceeded in the acidic pH range, and larger fibers were produced when organic solvents such as trifluoroethanol and acetonitrile were used at an acidic pH. Circular dichroism measurements of the spider peptide indicate that the peptide has a β‐sheet structure and that the formation of a β‐sheet structure is required for the spider peptide to undergo fiber formation.

Fiber Formation of a Synthetic Peptide Derived from Spider Dragline of Nephila Clavata

Spider dragline is a high performance biopolymer with exceptional mechanical properties. It is 5 times stronger than stainless wire, and high tensile strength and elasticity. The dragline is formed in the major ampullate gland of Nephila clavata and is composed of two major silk proteins, spidroin I and II. Our previous study suggested that the synthetic peptide derived from Nephila clavata forms large-sized fibers. However, the mechanism associated with spinning and the structure of the dragline silk protein remains to be studied in detail.To further investigate the relationship between structure and fiber formation, the fiber forming regions of spidroin were predicted, based on the hydrophobicity of individual amino acid residues. The candidate peptides were chemically synthesized by a solid-phase method, purified by reversed-phase HPLC, and the structures confirmed by MALD-TOF/MS analysis.The conditions for fiber formation for the candidate peptides were screened in a series of aqueous and organic solvents. Large-sized fibers were obtained when an organic solvent was used, under acidic conditions. However, the peptide was not able to form fibers under basic conditions.To obtain structural information on fiber formation, Circular Dichroism measurements of the synthetic spider peptides were performed. The results suggested that the formation of a β-sheet structure is required for fiber formation of the spider peptide.To characterize the peptide fibers, Dynamic laser light-scattering measurements were carried out under several conditions. The results indicated that the synthetic peptide formed a homogeneous oligomer at the initial time and moved to a large-sized oligomer at the later time.In conclusion, the fiber formation of the synthetic spider peptide occurs in organic solvents under acidic conditions and the synthetic peptide forms a homogeneous large-sized oligomer. The results will be discussed in this paper.