Abstract：
BACKGROUND: Caveolae are invaginated plasma membrane domains of 50-100 nm in diameter involved in many important physiological functions in eukaryotic cells. They are composed of different proteins, including the membrane-embedded caveolins and the peripheric cavins. Caveolin-1 has already been expressed in various expression systems (E. coli, insect cells, Toxoplasma gondii, cell-free system), generating intracellular caveolin-enriched vesicles in E. coli, insect cells and T. gondii. These systems helped to understand the protein insertion within the membrane and its oligomerization. There is still need for fundamental insights into the formation of specific domains on membrane, the deformation of a biological membrane driven by caveolin-1, the organization of a caveolar coat, and the requirement of specific lipids and proteins during the process. The aim of this study was to test whether the heterologously expressed caveolin-1β was able to induce the formation of intracellular vesicles within a Gram+ bacterium, Lactococcus lactis, since it displays a specific lipid composition different from E. coli and appears to emerge as a good alternative to E. coli for efficient overexpression of various membrane proteins.
RESULTS: Recombinant bacteria transformed with the plasmid pNZ-HTC coding for the canine isoform of caveolin-1β were shown to produce caveolin-1β, in its functional oligomeric form, at a high expression level unexpected for an eukaryotic membrane protein. Electron microscopy revealed several intracellular vesicles from 30 to 60 nm, a size comparable to E. coli h-caveolae, beneath the plasma membrane of the overexpressing bacteria, showing that caveolin-1β is sufficient to induce membrane vesiculation. Immunolabelling studies showed antibodies on such neo-formed intracellular vesicles, but none on plasma membrane. Density gradient fractionation allowed the correlation between detection of oligomers on Western blot and appearance of vesicles measurable by DLS, showing the requirement of caveolin-1β oligomerization for vesicle formation.
CONCLUSIONS: Lactococcus lactis cells can heterologously overexpress caveolin-1β, generating caveolin-1β enriched intracellular neo-formed vesicles. These vesicles might be useful for potential co-expression of membrane proteins of pharmaceutical interest for their simplified functional characterization.
RESULTS: Recombinant bacteria transformed with the plasmid pNZ-HTC coding for the canine isoform of caveolin-1β were shown to produce caveolin-1β, in its functional oligomeric form, at a high expression level unexpected for an eukaryotic membrane protein. Electron microscopy revealed several intracellular vesicles from 30 to 60 nm, a size comparable to E. coli h-caveolae, beneath the plasma membrane of the overexpressing bacteria, showing that caveolin-1β is sufficient to induce membrane vesiculation. Immunolabelling studies showed antibodies on such neo-formed intracellular vesicles, but none on plasma membrane. Density gradient fractionation allowed the correlation between detection of oligomers on Western blot and appearance of vesicles measurable by DLS, showing the requirement of caveolin-1β oligomerization for vesicle formation.
CONCLUSIONS: Lactococcus lactis cells can heterologously overexpress caveolin-1β, generating caveolin-1β enriched intracellular neo-formed vesicles. These vesicles might be useful for potential co-expression of membrane proteins of pharmaceutical interest for their simplified functional characterization.
摘要：
背景：Caveolae是直径为50-100nm的内陷质膜结构域，涉及真核细胞中许多重要的生理功能。它们由不同的蛋白质组成，包括膜包埋的窝蛋白和周围的窝蛋白。小窝蛋白-1已经在各种表达系统中表达(E.大肠杆菌昆虫细胞,弓形虫,无细胞系统)，在大肠杆菌中产生细胞内富含小窝蛋白的囊泡，昆虫细胞和弓形虫。这些系统有助于理解膜内的蛋白质插入及其寡聚化。仍然需要对膜上特定结构域形成的基本见解，由caveolin-1驱动的生物膜的变形，以及过程中对特定脂质和蛋白质的需求。这项研究的目的是测试异源表达的caveolin-1β是否能够诱导革兰氏细菌内细胞内囊泡的形成，乳酸乳球菌,因为它显示出不同于大肠杆菌的特定脂质组成,并且似乎是大肠杆菌有效过表达各种膜蛋白的良好替代品。
结果：用编码caveolin-1β的犬同工型的质粒pNZ-HTC转化的重组细菌显示产生caveolin-1β，以其功能性寡聚形式，在真核膜蛋白出乎意料的高表达水平。电子显微镜显示30至60nm的几个细胞内囊泡，与大肠杆菌h-caveolae相当的大小，在过表达细菌的质膜下，表明小窝蛋白-1β足以诱导膜囊泡形成。免疫标记研究表明，这种新形成的细胞内囊泡上有抗体，但质膜上没有.密度梯度分级允许在蛋白质印迹上检测寡聚体与通过DLS可测量的囊泡外观之间的相关性，显示囊泡形成需要小窝蛋白-1β寡聚化。
结论：乳酸乳球菌细胞可以异源过度表达小窝蛋白-1β，生成富含caveolin-1β的细胞内新形成的囊泡。这些囊泡可能可用于潜在的共表达具有药物意义的膜蛋白，以简化其功能表征。
结果：用编码caveolin-1β的犬同工型的质粒pNZ-HTC转化的重组细菌显示产生caveolin-1β，以其功能性寡聚形式，在真核膜蛋白出乎意料的高表达水平。电子显微镜显示30至60nm的几个细胞内囊泡，与大肠杆菌h-caveolae相当的大小，在过表达细菌的质膜下，表明小窝蛋白-1β足以诱导膜囊泡形成。免疫标记研究表明，这种新形成的细胞内囊泡上有抗体，但质膜上没有.密度梯度分级允许在蛋白质印迹上检测寡聚体与通过DLS可测量的囊泡外观之间的相关性，显示囊泡形成需要小窝蛋白-1β寡聚化。
结论：乳酸乳球菌细胞可以异源过度表达小窝蛋白-1β，生成富含caveolin-1β的细胞内新形成的囊泡。这些囊泡可能可用于潜在的共表达具有药物意义的膜蛋白，以简化其功能表征。
