Expression and Characterization of Recombinant C-Terminal Biotinylated Extracellular Domain of Human Receptor for Advanced Glycation End Products (hsRAGE) in Escherichia coli

Abstract

The receptor for advanced glycation end products (RAGE) is a multi-ligand receptor involved in the development of diabetic complications. Using an Escherichia coli expression system, we have successfully expressed and purified the C-terminal biotinylated extracellular domain of human RAGE (hsRAGE), which consists of three immunoglobulin-like domains carrying three putative disulfide bonds. Over 90% of hsRAGE was expressed in soluble form in trxB and gor mutant E. coli strain Origami (DE3). Most hsRAGE was biotinylated with a C-terminal AviTag, and stably immobilized onto matrix via streptavidin without any treatment. Immobilized hsRAGE without glycosylation recognized its ligands, such as AGEs. Biotinylated hsRAGE was also able to apply in the detection of AGEs on microtitre wells like antibodies used in enzyme-linked immunoassay. SPR analysis demonstrated that the dissociation constant (K(d)) of RAGE for AGE-BSA was 23.1 nM with the two-state reaction model, and 13.5 nM with the 1:1 binding model, comparable to those of RAGEs on cell surface. These results indicate that biotinylated hsRAGE must be useful not only in analysing RAGE-ligand interactions but also detect AGEs.

Co-expression of BirA with biotin bait achieves in vivo biotinylation of overexpressed stable N-glycosylated sRAGE in transgenic silkworms

“…In order to elucidate the interactions between RAGE and its ligands, the recombinant ligand recognition domain of RAGE has been produced 17–19 . Of the three domains of RAGE 1, 2 , the V and C1 domain represent an integrated structural unit, whereas the C2 domain is independent 14 .…”

“…Although sufficient amounts of active sRAGE have been produced using an E. coli system 19 , the sRAGE produced in E. coli is not stable and is degraded within 40 days. This lack of stability may be explained by the lack of N -glycosylation, a common post-translation modification that modulates both the physiological and biological properties of proteins 25, 26 .…”

“…Biotinylated sRAGE in E. coli was purified by sequential chromatography steps using Ni-NTA resin and streptavidin mutein matrix, as described previously 19 with modifications. Briefly, cleared lysate was mixed with Ni-NTA resin slurry for 15 min at 4 °C, and histidine-tagged proteins were eluted by FPLC with TBS containing 350 mM imidazole.…”

“…After the solution was discarded, the wells were washed three times with TBS containing 0.05% Tween 20 (TBS-T). The wells were then filled with 250 μL of nonprotein blocking buffer (Pierce) at room temperature for 2 h. After discarding the blocking solution, the wells were washed three times with TBS-T, 100 μL of AGE-BSA solution (0–8 μg/mL), prepared as previously described 19 , was applied to each well, and plates were incubated at room temperature for 2 h. The wells were then washed five times with TBS-T and incubated with 100 μL anti-BSA mouse monoclonal antibodies (Abcam; diluted 1:6000 with blocking buffer) for 1 h at room temperature. After washing the wells five times with TBS-T, the wells were incubated with 100 μL of anti-mouse IgG-HRP conjugate (Chemicon; diluted 1:5000 with blocking buffer) for 1 h at room temperature.…”

“…esRAGE is thought to function as a decoy receptor and bind AGEs without activating cellular signaling, whereas sRAGE blocks receptor activation in mouse models of various diseases 16, 17 . Currently, sRAGE has been expressed and purified from HEK293, Escherichia coli , Baculovirus, and yeast Pichia pastoris expression systems 2, 18, 19 . However, these expression systems do not yield sufficient amounts of sRAGE, mostly due to folding problems, protein instability, and high cost.…”