title: Preparation of metallochelating microbubbles and study on their site-specific interaction with rGFP-HisTag as a model protein.
abstract: The histidine-metallochelating 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-ligand complexes (e.g., streptavidine-biotin, glutathiontransferase-glutathion) 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 2.1-2.8 μm (the size range of 1-10 μm). The presence of large MB (8-10 μm) was marginal. Counting of MB revealed that the average amount of MB prepared of 10 mg of phospholipid equaled approximately 10(9) 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 °C was 3-7 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.
authors.affiliation: Department of Pharmacology, Toxicology and Immunotherapy, Veterinary Research Institute, Brno, Czech Republic.
authors.forename: Róbert
authors.initials: R
authors.lastname: Lukáč
authors.forename: Zuzana
authors.initials: Z
authors.lastname: Kauerová
authors.forename: Josef
authors.initials: J
authors.lastname: Mašek
authors.forename: Eliška
authors.initials: E
authors.lastname: Bartheldyová
authors.forename: Pavel
authors.initials: P
authors.lastname: Kulich
authors.forename: Štěpán
authors.initials: Š
authors.lastname: Koudelka
authors.forename: Zina
authors.initials: Z
authors.lastname: Korvasová
authors.forename: Jana
authors.initials: J
authors.lastname: Plocková
authors.forename: František
authors.initials: F
authors.lastname: Papoušek
authors.forename: František
authors.initials: F
authors.lastname: Kolář
authors.forename: Roland
authors.initials: R
authors.lastname: Schmidt
authors.forename: Jaroslav
authors.initials: J
authors.lastname: Turánek
mesh_terms: D000818:Animals; D001665:Binding Sites; D002614:Chelating Agents / Q000737:chemistry*; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D006639:Histidine; D008081:Liposomes / Q000378:metabolism*; D008670:Metals; D045423:Microbubbles; D008954:Models, Biological; D011485:Protein Binding; D011994:Recombinant Proteins
chemical_list: D002614:Chelating Agents; D008081:Liposomes; D008670:Metals; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D006639:Histidine