title: A Recombinant Human Anti-Platelet scFv Antibody Produced in Pichia pastoris for Atheroma Targeting.
abstract: Cells of the innate and adaptive immune system are key factors in the progression of atherosclerotic plaque, leading to plaque instability and rupture, potentially resulting in acute atherothrombotic events such as coronary artery disease, cerebrovascular disease and peripheral arterial disease. Here, we describe the cloning, expression, purification, and immunoreactivity assessment of a recombinant single-chain variable fragment (scFv) derived from a human anti-αIIbβ3 antibody (HuAb) selected to target atheromatous lesions for the presence of platelets. Indeed, platelets within atheroma plaques have been shown to play a role in inflammation, in platelet-leucocyte aggregates and in thrombi formation and might thus be considered relevant biomarkers of atherosclerotic progression. The DNA sequence that encodes the anti-αIIbβ3 TEG4 scFv previously obtained from a phage-display selection on activated platelets, was inserted into the eukaryote vector (pPICZαA) in fusion with a tag sequence encoding 2 cysteines useable for specific probes grafting experiments. The recombinant protein was expressed at high yields in Pichia pastoris (30 mg/L culture). The advantage of P. pastoris as an expression system is the production and secretion of recombinant proteins in the supernatant, ruling out the difficulties encountered when scFv are produced in the cytoplasm of bacteria (low yield, low solubility and reduced affinity). The improved conditions allowed for the recovery of highly purified and biologically active scFv fragments ready to be grafted in a site-directed way to nanoparticles for the imaging of atherosclerotic plaques involving inflammatory processes and thus at high risk of instability.
authors.affiliation: UMR5248, CBMN, Bordeaux-INP, Pessac, France.
authors.forename: Amelie
authors.initials: A
authors.lastname: Vallet-Courbin
authors.affiliation: Centre de Résonance Magnétique de Systèmes Biologiques, Centre Nationale de Recherche Scientifique et Université de Bordeaux, Bordeaux, France.
authors.forename: Mélusine
authors.initials: M
authors.lastname: Larivière
authors.affiliation: UMR5248, CBMN, Bordeaux-INP, Pessac, France.
authors.forename: Agnès
authors.initials: A
authors.lastname: Hocquellet
authors.affiliation: Centre de Résonance Magnétique de Systèmes Biologiques, Centre Nationale de Recherche Scientifique et Université de Bordeaux, Bordeaux, France.
authors.forename: Audrey
authors.initials: A
authors.lastname: Hemadou
authors.affiliation: UMR5248, CBMN, Bordeaux-INP, Pessac, France.
authors.forename: Sarjapura-Nagaraja
authors.initials: SN
authors.lastname: Parimala
authors.affiliation: Centre de Résonance Magnétique de Systèmes Biologiques, Centre Nationale de Recherche Scientifique et Université de Bordeaux, Bordeaux, France.
authors.forename: Jeanny
authors.initials: J
authors.lastname: Laroche-Traineau
authors.affiliation: UMR5248, CBMN, Bordeaux-INP, Pessac, France.
authors.forename: Xavier
authors.initials: X
authors.lastname: Santarelli
authors.affiliation: Centre de Résonance Magnétique de Systèmes Biologiques, Centre Nationale de Recherche Scientifique et Université de Bordeaux, Bordeaux, France.
authors.forename: Gisèle
authors.initials: G
authors.lastname: Clofent-Sanchez
authors.affiliation: Centre de Résonance Magnétique de Systèmes Biologiques, Centre Nationale de Recherche Scientifique et Université de Bordeaux, Bordeaux, France.
authors.forename: Marie-Josée
authors.initials: MJ
authors.lastname: Jacobin-Valat
authors.affiliation: UMR5248, CBMN, Bordeaux-INP, Pessac, France.
authors.forename: Abdelmajid
authors.initials: A
authors.lastname: Noubhani
mesh_terms: D001792:Blood Platelets / Q000276:immunology*; D061505:Cell Surface Display Techniques; D003001:Cloning, Molecular; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D007249:Inflammation / Q000188:drug therapy* / Q000276:immunology; D010843:Pichia / Q000235:genetics; D058226:Plaque, Atherosclerotic / Q000188:drug therapy* / Q000276:immunology; D010974:Platelet Aggregation / Q000276:immunology; D011994:Recombinant Proteins / Q000276:immunology* / Q000627:therapeutic use; D057127:Single-Chain Antibodies / Q000276:immunology* / Q000627:therapeutic use
chemical_list: D011994:Recombinant Proteins; D057127:Single-Chain Antibodies