Preparation of Metallochelating Microbubbles and Study on Their Site-Specific Interaction with rGFP-HisTag as a Model Protein

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• 作者：Ro虂bert Luka虂č ; Zuzana Kauerova虂 ; Josef Mas虒ek ; Elis虒ka Bartheldyova虂 ; Pavel Kulich ; S虒te虒pa虂n Koudelka ; Zina Korvasova虂 ; Jana Plockova虂 ; Frantis虒ek Papous虒ek ; Frantis虒ek Kola虂ř ; Roland Schmidt ; Jaroslav Tura虂nek
• 刊名：Langmuir
• 出版年：2011
• 出版时间：April 19, 2011
• 年：2011
• 卷：27
• 期：8
• 页码：4829-4837
• 全文大小：1002K
• 年卷期：v.27,no.8(April 19, 2011)
• ISSN：1520-5827

文摘

The histidine鈭抦etallochelating lipid complex is one of the smallest high affinity binding units used as tools for rapid noncovalent binding of histidine tagged molecules, especially recombinant proteins. The advantage of metallochelating complex over protein鈭抣igand complexes (e.g., streptavidine鈭抌iotin, glutathiontransferase鈭抔lutathion) consists in its very low immunogenicity, if any. This concept for the construction of surface-modified metallochelating microbubbles was proved with recombinant green fluorescent protein (rGFP) containing 6His-tag. This protein is easy to be detected by various fluorescence techniques as flow cytometry and confocal microscopy. Microbubbles (MB) composed of DPPC with various contents of metallochelating lipid DOGS-NTA-Ni were prepared by intensive shaking of the liposome suspension under the atmosphere of sulfur hexafluoride. For this purpose, the instrument 3M ESPE CapMix was used. Various techniques (static light scattering, flow cytometry, and optical microscopy) were compared and used for the measurements of the size distribution of MB. All three methods demonstrated that the prepared MB were homogeneous in their size, and the mean diameter of the MB in various batches was within the range of 2.1鈭?.8 渭m (the size range of 1鈭?0 渭m). The presence of large MB (8鈭?0 渭m) was marginal. Counting of MB revealed that the average amount of MB prepared of 10 mg of phospholipid equaled approximately 10⁹ MB/mL. Lyophilized MB were prepared with saccharose as a cryoprotectant. These MB were shown to be stable both in vitro (the estimated half-live of the MB in bovine serum at 37 掳C was 3鈭? min) and in vivo (mouse). The stability of the MB was affected by molar content of DOGS-NTA-Ni. DPPC-based metallochelating MB provided a clear and very contrast image of the ventricular cavity soon after the injection. Site selective and stable binding of rGFP-HisTag (as a model of His-tagged protein) onto the surface of metallochelating MB was demonstrated by confocal microscopy.