Recombinant expression and characterization of an epididymis-specific antimicrobial peptide BIN1b/SPAG11E.
BIN1b was reported as an epididymis-specific beta-defensin antimicrobial peptide. In this paper, the recombinant BIN1b was expressed and purified by fusing with GB1-His tag. The size-exclusion gel filtration experiment indicated that the fusion protein GB1-BIN1b formed multimers at pH 7.4, and existed as monomer at pH 4.5. The oligomerization of GB1-BIN1b was only related to pH value, neither to NaCl concentration nor protein concentration. Far-UV circular dichroism (CD) spectra also showed the fusion protein had more ordered secondary structures at pH 4.5 than at pH 7.4, as a negative peak appeared around 218 nm indicative of typical beta-sheet. The 2D (15)N-(1)H heteronuclear single-quantum coherence (HSQC) spectra suggested that the fusion protein adopted a compact three-dimensional structure at pH 4.5. Colony forming unit (CFU) inhibition assay demonstrated that 25 microM fusion protein at pH 7.4 had an antimicrobial activity of 40% against E. coli K(12)D(31), which might imply the fusion protein functions as multimeric states. In conclusion, the GB1 fusion partner helps BIN1b form a stable homogenous conformation to facilitate subsequent structural determination without a significant effect on the antimicrobial activity.
Guo
Chenyun
C
Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 201203, PR China.
Diao
Hua
H
Lian
Yandong
Y
Yu
Heguo
H
Gao
Hongchang
H
Zhang
Yonglian
Y
Lin
Donghai
D
D000595:Amino Acid Sequence; D023181:Antimicrobial Cationic Peptides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D015169:Colony Count, Microbial; D004822:Epididymis / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D023083:beta-Defensins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D023181:Antimicrobial Cationic Peptides; D011993:Recombinant Fusion Proteins; D023083:beta-Defensins