title: GFP-SCFV: expression and possible applications as a tool for experimental investigations of atherosclerosis.
abstract: Experimental studies on atherosclerosis are crucial for investigating its pathophysiology, defining new therapeutic targets, and developing new drugs and diagnostic tools. Thus, many imaging markers have been developed and introduced in experimental studies. The main advantage of these new tools is that they allow the noninvasive diagnosis of atherosclerotic vascular disease. Here, we describe the cloning, expression, purification, and stabilization of a chimeric protein specifically designed to probe cells and tissues for the presence of LDL(-), a relevant marker of atherosclerosis. The DNA sequence that encodes the anti-LDL(-) scFv, previously obtained from a hybridoma secreting an anti-LDL(-) monoclonal antibody, was inserted into the bacterial vector pET-28a(+) in tandem with a DNA sequence encoding GFP. The recombinant protein was expressed in high yields in E. coli as inclusion bodies. The applicability of GFP-scFv was assessed by ELISA, which determined its affinity for LDL(-) and confocal microscopy, that showed macrophage uptake of the protein along with LDL(-). In conclusion, our data suggest that the anti-LDL(-) GFP-scFv chimeric protein could be useful in studies on atherogenesis as well as for developing diagnostic tools for atherosclerosis.
authors.affiliation: Dept. of Clinical and Toxicological Analyses, Faculty of Pharmaceutical Sciences, University of Sao Paulo, Brazil.
authors.forename: Tanize do Espirito Santo
authors.initials: Tdo E
authors.lastname: Faulin
authors.forename: Daniel Ferreira
authors.initials: DF
authors.lastname: Guilherme
authors.forename: Aldacilene Souza
authors.initials: AS
authors.lastname: Silva
authors.forename: Dulcineia Saes Parra
authors.initials: DS
authors.lastname: Abdalla
authors.forename: Vitor Renaux
authors.initials: VR
authors.lastname: Hering
authors.forename: Mario José
authors.initials: MJ
authors.lastname: Politi
authors.forename: Andrea Queiroz
authors.initials: AQ
authors.lastname: Maranhão
mesh_terms: D000818:Animals; D050197:Atherosclerosis / Q000378:metabolism; D015415:Biomarkers / Q000378:metabolism*; D045744:Cell Line, Tumor; D004705:Endocytosis; D004926:Escherichia coli / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000378:metabolism* / Q000493:pharmacokinetics; D008077:Lipoproteins, LDL / Q000378:metabolism*; D051379:Mice; D057054:Molecular Imaging / Q000379:methods; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism* / Q000493:pharmacokinetics; D057127:Single-Chain Antibodies / Q000737:chemistry / Q000378:metabolism* / Q000493:pharmacokinetics
chemical_list: D015415:Biomarkers; D008077:Lipoproteins, LDL; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies; C099252:oxidized low density lipoprotein; D049452:Green Fluorescent Proteins
keywords: atherosclerosis; electronegative LDL; fluobodies; recombinant protein; refolding