Use of the VC1 domain of human RAGE for the affinity purification of AGEs

Objectives: The aim of this work has been the set-up of a system for the capture of AGEs. A recombinant form of the integrated V and C1 domains of hRAGE [1] responsible for AGE binding was immobilized on solid matrices and tested in pull-down assays. Methodology: The VC1 domain, with two additional affinity tags at the C-terminal end, was produced in Pichia pastoris that previously proved to be a suitable host for the secretion of glycosylated and soluble VC1 [2]. The fusion protein was immobilized on magnetic beads and the derived VC1-resin was used for investigating its in vitro binding properties. Results: The VC1-resin captured high MW AGE-BSA (obtained from incubation of BSA with sugars) and did not bind unmodified BSA. Mass spectrometry (MS) analyses of BSA-ribose products, eluted from the VC1-resin, identified the location and a series of AGE modifications that were previously described as typical of BSA-ribose [3]. The VC1-resin also specifically captured BSA-ribose in a simulated complex matrix such as milk. Conclusion: We developed an affinity purification method for AGEs. This method, combined with MS, is prospectively useful for the study and identification of AGEs in clinical samples or food. Patent application number: PCT/IB2016/052391