Cell surface display of chimeric glycoproteins via the S-layer of Paenibacillus alvei.
Cell surface display of chimeric glycoproteins via the S-layer of Paenibacillus alvei.
复制标题
DOI：
10.1016/j.carres.2010.04.010
复制
发表时间：
2010-07-02
影响因子：
3.1
通讯作者：
Schaeffer, Christina
中科院分区：
文献类型：
--
作者：
Zarschler, Kristof;Janesch, Bettina;Kainz, Birgit;Ristl, Robin;Messner, Paul;Schaeffer, Christina
关键词：
革兰氏阳性嗜温菌Paenibacillus alvei CCM 2051 T具有由单一类型的O-糖蛋白种类组成的具有斜晶格对称性的二维晶体蛋白表面层（S-层）。在此，我们描述了一种基于S层糖蛋白自组装系统的纳米图案化的肽和聚糖表位在体内细胞表面共展示的策略。相应结构基因spaA的开放阅读框编码983个氨基酸的蛋白质，包括24个氨基酸的信号肽。成熟的S-层蛋白的理论分子量为105.95 kDa，计算的pI为5.83。它在N-末端含有三个S-层同源结构域，这些结构域参与糖蛋白通过非经典的乙酰化次级细胞壁聚合物锚定到细胞壁的肽聚糖层。对于这种聚合物，几个推定的生物合成酶被确定的spaA基因的上游。为了在体内细胞表面展示，六聚组氨酸标签和增强的绿色荧光蛋白，分别被选择性地融合到SpaA的C-末端。免疫印迹分析、免疫荧光染色和荧光显微镜观察显示融合表位在蜂房毕赤酵母CCM 2051 T细胞表面的S层糖蛋白基质中得到有效表达和成功展示。相反，当糖基化缺陷的wsfP突变体的S-层用作展示基质时，仅展示非糖基化的嵌合SpaA蛋白。
The Gram-positive, mesophilic bacterium Paenibacillus alvei CCM 2051T possesses a two-dimensional crystalline protein surface layer (S-layer) with oblique lattice symmetry composed of a single type of O-glycoprotein species. Herein, we describe a strategy for nanopatterned in vivo cell surface co-display of peptide and glycan epitopes based on this S-layer glycoprotein self-assembly system. The open reading frame of the corresponding structural gene spaA codes for a protein of 983 amino acids, including a signal peptide of 24 amino acids. The mature S-layer protein has a theoretical molecular mass of 105.95 kDa and a calculated pI of 5.83. It contains three S-layer homology domains at the N-terminus that are involved in anchoring of the glycoprotein via a non-classical, pyruvylated secondary cell wall polymer to the peptidoglycan layer of the cell wall. For this polymer, several putative biosynthesis enzymes were identified upstream of the spaA gene. For in vivo cell surface display, the hexahistidine tag and the enhanced green fluorescent protein, respectively, were translationally fused to the C-terminus of SpaA. Immunoblot analysis, immunofluorescence staining, and fluorescence microscopy revealed that the fused epitopes were efficiently expressed and successfully displayed via the S-layer glycoprotein matrix on the surface of P. alvei CCM 2051T cells. In contrast, exclusively non-glycosylated chimeric SpaA proteins were displayed, when the S-layer of the glycosylation-deficient wsfP mutant was used as a display matrix.
登录
查看更多内容
影响因子：
6.8
作者：
Gai, S. Annie;Wittrup, K. Dane
通讯作者：
Wittrup, K. Dane
影响因子：
3.2
作者：
Bingle, WH;Nomellini, JF;Smit, J
通讯作者：
Smit, J
影响因子：
4
作者：
Mesnage, S;Tosi-Couture, E;Fouet, A
通讯作者：
Fouet, A
影响因子：
3.2
作者：
HARTMANN, E;MESSNER, P;KONIG, H
通讯作者：
KONIG, H
影响因子：
3.2
作者：
Bingle, WH;Nomellini, JF;Smit, J
通讯作者：
Smit, J