Purification, Characterization And Crystallization Of A Novel Antimicrobial Peptide From Mung Bean

A novel 9-kDa nonspecific lipid transfer protein(nsLTP) with antimicrobial activity wasisolated from mung bean (Phaseolus mungo) seeds, which was denominated as pm-nsLTP1. Thepurification procedure involved ammonium sulfate precipitation, ion exchange chromatographyon CM-sephadex C-50 column, High Performance Liquid Chromatography (HPLC) on POROSHS-20 column. The active peptide(pm-nsLTP1)was a monomer with intramolecular disulfide bridges, aswas estimated from mass spectrometry, SDS-PAGE electrophoresis, N-terminal amino acidsequence analysis. pm-nsLTP1 was tested to have strong growth inhibitory activity in vitroagainst plant pathogenic fungi Fusarium solani, Fusarium oxysporum, Pythium aphanidermatum,and Sclerotium rolfsii, furthermore it also showed inhibitory activity against Gram-positivebacterial pathogen Staphylococcus aureus with minimum inhibitory concentration of 0.06mmol/ml. The lipid binding of pm-nsLTP1 was investigated in aqueous solution by fluorescencespectroscopy using lyso-12 as a substrate, and the result manifested the relative increase offluorescence intensity saturated around 120% at a lipid/peptide ration (Ri) equal to 8. Thepeptide partial N-terminal amino acid sequence was identified as MTCGQVQGNLAQCIGFLEKGG-21, which displayed a high degree of homology to other antifungal peptides isolated fromplants by analysis using computer soft CLUSTAL W. Through the determination of scanning electron microscope, it was observed thatpm-nsLTP1 induced lysis of the bacterial pathogen Staphylococcus aureus, and resulted in walldisruption, release of cell sap from cell walls and cytoplasm leakage. A probable mechanism wasproposed that the interplay of molecule structural characteristics, such as its binding structureand its hydrophobic hole structure, would have attributed to its activity in antimicrobe. A crystallization strategy designed for pm-nsLTP1 according to the Crystal Screen thatincluded thirty eight sorts of crystal conditions. Among them we found some crystals growedunder three conditions, namely No.3, No.6, No.9. The No.6 condition,namely 30% PEG4000,0.1mol/L Tris-HCl, pH8.5, 0.2mol/L MgCl2, was further optimized, to finally obtain a crystal tobe X –ray diffraction. The result showed that the crystal cell parameters was identical as bond 2lengths of a=8.737?, b=9.341 ?, c=8.745 ?, and as bond angles of α=90.113℃,β=90.169℃,γ=90.443℃. Crystal growth studies of the protein is under way.