Production of bioactive recombinant rat soluble receptor for advanced glycation end products (rrsRAGE) in Pichia...

Soluble receptor for advanced glycation end products (sRAGE), a natural inhibitor of RAGE, is considered to be a putative therapeutic molecule for a variety of diseases and a biomarker for certain conditions. To further study the function of sRAGE, recombinant rat sRAGE (rrsRAGE) was expressed and produced in a eukaryotic system. The open reading frame of rat sRAGE was cloned downstream of the methanol-inducible alcohol oxidase promoter of pPICZαA vector, and Pichia pastoris strain X-33 was used as the host strain. The expression of rrsRAGE was achieved by fermentation in a 15-L bioreactor and the resulting fermentation broth was subjected to purification on a cation exchange chromatography column. The purification of rrsRAGE reached 95% after size exclusion chromatography(SEC). The bioactivity of the purified protein was confirmed in a SH-SY5Y cell proliferation assay. The biological function of the purified rrsRAGE protein rat CCl₄-induced model was then examined. Treatment with rrsRAGE resulted in significantly lower liver fibrosis and lower serum level of ALT, suggesting that sRAGE prevent liver from injury and fibrosis. In conclusion, we achieved high-efficiency production of bioactive rrsRAGE in P. pastoris.