Expression Of Human Soluble Receptor For Advanced Glycation End Product In Pichia Pastoris And Its Fermentation Technic Research

Receptor for advanced glycation end product (RAGE) is a transmembrane protein.The association of RAGE and advanced glycation end products (AGEs) can lead to a variety of pathological effects. Soluble receptor for advanced glycation end product (sRAGE) is the extracellular section of RAGE.As a competitive antagonist of RAGE,sRAGE can specifically bind with AGEs and, sequentially,block the pathological effects caused by the cytosol domain of RAGE of AGE-RAGE signaling system.Objective:To study the expression of human soluble receptor for advanced glycation end product (hsRAGE) using the Pichia pastoris expression system,optimize fermentation conditions of the recombinant Pichia pastoris engineering strain and study the activity of expressed product in vitro.This work will pave the way for further research and application of hsRAGE.Methods:The hsRAGE cDNA was inserted into Pichia pastoris constitutive expression vector, pGAPZaA,the positive colonies were selected by YPD culture plate containing Zeocin.Under the control of the promoter GAP (glyceraldehydes 3 phosphate dehydrogenase),hsRAGE was expressed constitutively.The primary expression conditions experiments were carried out by means of shake flask.Then,the optimization of fermentation technics was carried out in fermentor.The expressed product was purified by Ni-NTA affinity chromatography and identified by Western blot and enzyme-linked immunosorbent assay (ELISA) blockade experiments in the endothelial cells.The effect of hsRAGE on the NF-κB signal transduction pathway of endothelial cells was detected by Western blot.Results:hsRAGE protein was successfully expressed by Pichia Pastroris constitutive expression system, the protein with a molecular weight of about 45kDa.The optimum fermentation of constitutive expression were as follow:30℃,pH6.0,the expression level of hsRAGE reached its peak at 60 hours.The expression level of hsRAGE was 58.5mg/L.This protein could be specifically immunoprecipitated by mouse monoclonal antibodies against human sRAGE. After affinity purification,the protein displays the effect of binding the AGE and blocking AGE-induced nuclear factor NF-κB activation in the endothelial cells.This study could be available for the large-scale industrial production and play an important role in the biological and clinical research of hsRAGE.Conclusion:1. The Pichia pastoris constitutive expression system is favourable for expression of hsRAGE.2. The expression level of hsRAGE was remarkably increase by optimization of fermentation technics.3. The recombinant hsRAGE expressed by Pichia pastoris has significant biological activity and of brilliant prospect in treat with the RAGE related diseases.4. The successful expression of hsRAGE in Pichia Pastoris and optimization of fermentation technics lay the foundations for the further study and large-scale production of hsRAGE.