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. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1206–1213, 2014

One-Step Ultrasensitive Bioluminescent Enzyme Immunoassay Based on Nanobody/Nanoluciferase Fusion for Detection of Aflatoxin B1 in Cereal

“…AP-nanobody fusions have also been utilized for the detection of small molecules, such as ochratoxin A, tetrabromobisphenol A, and parathion . Another group of reporters are fluorescent proteins (i.e., GFP and RFP), which have been used for construction of fusion proteins with antibody fragments. − The one-step immunoassay based on antibody–reporter fusions can reduce long assay times, which is a drawback for traditional immunoassays requiring secondary or tertiary antibody enzyme conjugates. Another important group of photoproteins are luciferases.…”

Production and applications of fluorobody from redox-engineered Escherichia coli

“…6b ). Similar application of scFv fusion to C-terminus of GFP for cell-based analysis of LDL uptake mechanisms using GFP-scFv against LDL has been reported (Cardinale et al 2004 ; Faulin et al 2014 ).…”

Proinflammatory Action of a New Electronegative Low-Density Lipoprotein Epitope

“…Furthermore, passive immunization with these anti-LDL (−) antibodies has been shown to be atheroprotective in Ldlr −/− mice [20]. Following this work, other studies have produced an anti-LDL (−) single-chain fragment variable (scFv) of the 2C7 monoclonal antibody that has potential applications in atherosclerosis therapy and diagnosis [21,22,23]. However, the molecular properties of immunodominant proinflammatory epitopes of LDL (−) remain unknown.…”