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首页> 外文期刊>Applied Microbiology >Production, Secretion, and Cell Surface Display of Recombinant Sporosarcina ureae S-Layer Fusion Proteins in Bacillus megaterium
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Production, Secretion, and Cell Surface Display of Recombinant Sporosarcina ureae S-Layer Fusion Proteins in Bacillus megaterium

机译：巨大芽孢杆菌中重组孢子脲脲S-层融合蛋白的产生，分泌和细胞表面展示

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Monomolecular crystalline bacterial cell surface layers (S-layers) have broad application potential in nanobiotechnology due to their ability to generate functional supramolecular structures. Here, we report that Bacillus megaterium is an excellent host organism for the heterologous expression and efficient secretion of hemagglutinin (HA) epitope-tagged versions of the S-layer protein SslA from Sporosarcina ureae ATCC 13881. Three chimeric proteins were constructed, comprising the precursor, C-terminally truncated, and N- and C-terminally truncated forms of the S-layer SslA protein tagged with the human influenza hemagglutinin epitope. For secretion of fusion proteins, the open reading frames were cloned into the Escherichia coli-Bacillus megaterium shuttle vector pHIS1525. After transformation of the respective plasmids into Bacillus megaterium protoplasts, the recombinant genes were successfully expressed and the proteins were secreted into the growth medium. The isolated S-layer proteins are able to assemble in vitro into highly ordered, crystalline, sheetlike structures with the fused HA tag accessible to antibody. We further show by fluorescent labeling that the secreted S-layer fusion proteins are also clustered on the cell envelope of Bacillus megaterium , indicating that the cell surface can serve in vivo as a nucleation point for crystallization. Thus, this system can be used as a display system that allows the dense and periodic presentation of S-layer proteins or the fused tags.
机译：单分子晶体细菌细胞表面层（S层）由于其产生功能性超分子结构的能力而在纳米生物技术中具有广泛的应用潜力。在这里，我们报告的巨大芽孢杆菌是异源表达和有效分泌的血凝素（HA）表位标记版本的S层蛋白SslA从Sporosarcina尿素ATCC 13881的优秀宿主生物。构建了三个嵌合蛋白，包括前体，以人流感血凝素表位标记的S层SslA蛋白的C末端，C末端截短以及N末端和C末端截短的形式。为了分泌融合蛋白，将开放阅读框克隆到大肠杆菌-巨大芽孢杆菌穿梭载体pHIS1525中。将各个质粒转化成巨大芽孢杆菌原生质体后，重组基因被成功表达，蛋白质被分泌到生长培养基中。分离出的S层蛋白能够在体外组装成高度有序的，晶体状的片状结构，其融合的HA标签可被抗体使用。我们进一步通过荧光标记显示，分泌的S层融合蛋白也聚集在巨大芽孢杆菌的细胞包膜上，表明细胞表面可以在体内充当结晶的成核点。因此，该系统可用作允许S层蛋白或融合标签的密集且周期性呈现的展示系统。

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