Ludwig-Maximilians-Universität München, Dept. Biologie I, Bereich Mikrobiologie, Biozentrum der LMU, Großhaderner Str. 2-4, D-82152 Martinsried, Germany.
Appl Environ Microbiol. 2011 Sep;77(17):6165-71. doi: 10.1128/AEM.05282-11. Epub 2011 Jul 15.
Numerous applications of conventional and biogenic magnetic nanoparticles (MNPs), such as in diagnostics, immunomagnetic separations, and magnetic cell labeling, require the immobilization of antibodies. This is usually accomplished by chemical conjugation, which, however, has several disadvantages, such as poor efficiency and the need for coupling chemistry. Here, we describe a novel strategy to display a functional camelid antibody fragment (nanobody) from an alpaca (Lama pacos) on the surface of bacterial biogenic magnetic nanoparticles (magnetosomes). Magnetosome-specific expression of a red fluorescent protein (RFP)-binding nanobody (RBP) in vivo was accomplished by genetic fusion of RBP to the magnetosome protein MamC in the magnetite-synthesizing bacterium Magnetospirillum gryphiswaldense. We demonstrate that isolated magnetosomes expressing MamC-RBP efficiently recognize and bind their antigen in vitro and can be used for immunoprecipitation of RFP-tagged proteins and their interaction partners from cell extracts. In addition, we show that coexpression of monomeric RFP (mRFP or its variant mCherry) and MamC-RBP results in intracellular recognition and magnetosome recruitment of RFP within living bacteria. The intracellular expression of a functional nanobody targeted to a specific bacterial compartment opens new possibilities for in vivo synthesis of MNP-immobilized nanobodies. Moreover, intracellular nanotraps can be generated to manipulate bacterial structures in live cells.
许多传统和生物磁纳米粒子（MNPs）的应用，如诊断、免疫磁分离和磁性细胞标记，都需要抗体的固定化。这通常通过化学偶联来实现，但这种方法存在几个缺点，例如效率低和需要偶联化学。在这里，我们描述了一种将骆驼科抗体片段（纳米抗体）从羊驼（Lama pacos）展示在细菌生物磁纳米粒子（磁小体）表面的新策略。通过将 RFP 结合纳米抗体（RBP）与产磁铁矿细菌 Magnetospirillum gryphiswaldense 中的磁小体蛋白 MamC 进行基因融合，在体内实现了 RFP 结合纳米抗体在磁小体中的特异性表达。我们证明了表达 MamC-RBP 的分离磁小体能够在体外有效地识别和结合它们的抗原，并可用于从细胞提取物中免疫沉淀 RFP 标记的蛋白质及其相互作用伙伴。此外，我们还表明，单体 RFP（mRFP 或其变体 mCherry）和 MamC-RBP 的共表达导致 RFP 在活细菌内的细胞内识别和磁小体募集。针对特定细菌隔室的功能性纳米抗体的细胞内表达为体内合成固定化纳米抗体开辟了新的可能性。此外，可以生成细胞内纳米陷阱来操纵活细胞中的细菌结构。
