Kari Keinänen

Publications (3)0 Total impact

• Article: Use of proteoliposomes to generate phage antibodies against native AMPA receptor

  Lene K. Jespersen, Arja Kuusinen, Adelina Orellana, Kari Keinänen, Jan Engberg
  [show abstract] [hide abstract]
  ABSTRACT: To isolate antibodies against ionotropic glutamate receptors (GluRs), we prepared a phage antibody library from mice immunized with proteoliposomes containing purified α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), a selective GluRD receptor. Specific binders were selected by repeated rounds of affinity panning against immobilized GluRD liposomes. Using this approach, we obtained a panel of high-affinity antibody fragments that immunoprecipitated both recombinant and native GluRD receptors, but not GluR6, a kainate receptor subunit with a 40% sequence similarity. The antibody fragments showed subunit selectivity, some being strictly specific for GluRD, whereas others also recognized the GluRB and GluRC but not GluRA subunits. Further experiments indicated that the epitopes recognized were conformational in nature and reside in the N-terminal extracellular 400-residue X domain of GluRD. Our results suggest that proteoliposomes, in combination with phage display technology, provide an effective tool for the generation of high-affinity conformation-sensitive monoclonal antibodies against predetermined membrane proteins.
  European Journal of Biochemistry. 02/2000; 267(5):1382 - 1389.
• Article: Europium chelate-loaded liposomes: a tool for the study of binding and integrity of liposomes

  Adelina Orellana, Marja-Leena Laukkanen, Kari Keinänen
  [show abstract] [hide abstract]
  ABSTRACT: Using the biotin-streptavidin interaction as a model, we investigated the suitability of lanthanide chelates as encapsulated liposomal labels in liposome-based binding assays. Large unilamellar phospholipid:cholesterol liposomes containing europium-DTPA chelate and biotinylated phosphatidylethanolamine were prepared by detergent dialysis. The resulting Eu-liposomes (⊘120 nm) bound specifically to streptavidin in microtiter wells as measured by time-resolved fluorometric assay (TRF). The intensity of fluorescence released from the bound liposomes was dependent on the concentration of biotin in the liposome membrane, the concentration of europium entrapped in the liposomes, the incubation time and the amount of liposomes used in the assay. The sensitivity of the TRF assay allowed the detection of binding of attomole quantities of liposomes. The streptavidin-immobilised liposomes subjected to porcine pancreatic phospholipase A2 (EC 3.1.1.4) and detergents displayed a dose-dependent release of the encapsulated europium. Lanthanide-chelate-liposomes should prove useful for studies addressing binding and stability of liposomes.
  Biochimica et Biophysica Acta (BBA) - Biomembranes.
• Article: Mimicking rubella virus particles by using recombinant envelope glycoproteins and liposomes

  Adelina Orellana, David Mottershead, Inge van der Linden, Kari Keinänen, Christian Oker-Blom
  [show abstract] [hide abstract]
  ABSTRACT: The envelope glycoproteins E1 and E2 of rubella virus (RV) were engineered to display the FLAG epitope tag and a polyhistidine tag, at their amino and carboxy termini, respectively. These modified envelope proteins were produced in Sf9 insect cells utilizing baculovirus expression vectors, the E1 and E2 vectors giving rise to protein products of about 58 and 42 kDa, respectively. The recombinant proteins were purified by immobilized metal-ion affinity chromatography and reconstituted into liposomes via their hydrophobic transmembrane anchors. The liposomes were prepared by detergent dialysis in the presence of europium-DTPA chelate, enabling the subsequent measurement of the binding of the resultant proteoliposomes to the antibodies by time resolved fluorescence. RV mimicking proteoliposomes were recognized by antibodies specific for the E1 and E2 proteins, as well as the FLAG epitope tag. This type of virosome may prove useful for studies on the basic biological events of an RV infection or as diagnostic reagents.
  Journal of Biotechnology.