Guo Chenyun, Diao Hua, Lian Yandong, Yu Heguo, Gao Hongchang, Zhang Yonglian, Lin Donghai
Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 201203, PR China.
J Biotechnol. 2009 Jan 1;139(1):33-7. doi: 10.1016/j.jbiotec.2008.10.003. Epub 2008 Oct 21.
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.
BIN1b被报道为一种附睾特异性β-防御素抗菌肽。在本文中，重组BIN1b通过与GB1-His标签融合进行表达和纯化。尺寸排阻凝胶过滤实验表明，融合蛋白GB1-BIN1b在pH 7.4时形成多聚体，在pH 4.5时以单体形式存在。GB1-BIN1b的寡聚化仅与pH值有关，与NaCl浓度和蛋白质浓度均无关。远紫外圆二色（CD）光谱也显示，融合蛋白在pH 4.5时比在pH 7.4时具有更多有序的二级结构，因为在218 nm左右出现了一个负峰，表明存在典型的β-折叠。二维（15）N-（1）H异核单量子相干（HSQC）光谱表明，融合蛋白在pH 4.5时呈现紧密的三维结构。菌落形成单位（CFU）抑制试验表明，pH 7.4时25 μM的融合蛋白对大肠杆菌K（12）D（31）具有40%的抗菌活性，这可能意味着融合蛋白以多聚体状态发挥作用。总之，GB1融合伴侣有助于BIN1b形成稳定的同源构象，便于后续的结构测定，且对抗菌活性没有显著影响。
