Expression And Purification Of Aldose Reductase Related Diabetic Complication

Diabetes mellitus (DM) is a serious disease with severe complications such as cataract, neuropathy, nephropathy, and retinopathy. With the development of diabetes, approximately 73.2% patients are prone to accompany one or more diabetic complications.The survival rate of chronic diabetic patients remains very low despite improvement of diagnostics and therapy over the past decade. Polyol pathway plays an important role during the development of diabetic complications. Aldose reductase (AR), the first and rate-limiting enzyme in polyol pathway of sugar metabolism, has been implicated with diabetic complications. AR Inhibitors (ARIs), the first choice drugs in the treatment of diabetic complications, are effective to prevent, delay or arrest the development of diabetic complications in animal models and in clinical trials. Now the existing drugs (ARIs, like Tolrestat) have some side effects,so it is important to look for new aldose reducatse inhibitor with more safety and better effects.The aim of this study was to clon and express human aldose reductase gene (hAR), and achieved the aldose reductase with biological activity. And it would be the foundation for screening the new aldose reductase inhibitors from national drugs in vitro.Human aldose reductase(hAR) mRNA was detected in human liver cancer SMMC7721 stimulated for 3 days with D-glucose (50mM) by real-time PCR. Aldose reductase cDNA was generated by reverse transcription-PCR (RT-PCR) from the isolated total RNA in SMMC7721 cells. The PCR product was cloned into E.coli expression vector pET22b(+) to create a recombinant plasmid with 6×His Tag , which was named pET22b(+)-AR. The AR-(His)6 fusion protein was induced and expressed in E.coli BL21(DE3) by 0.1mM IPTG. AR-(His)6 was purified by Ni-NTA affinity chromotagraphy and revealed by Western-blotting analysis anti-(His)6 Tag monoclonal antibody or anti-AR monoclonal antibody. Finally the purified AR-(His)6 activity enzyme was measured by AR assay with ultraviolet spectrophotometry.In conclusion: The studies successfully constructed the prokaryotic recombinant plasmids pET22b(+)-AR , expressed and purified the aldose reductase 6×His Tag fusion proteins. SDS-PAGE revealed the protein with apparent molecular weight of approximately 36 kDa. Western-blotting analysis showed the fusion protein against anti-6×His monoclonal antibody or anti-AR monoclonal antibody.The results of this study may represent a basis for development of potential drug in preventing diabetic complications. To screen highly efficient and selective aldose reductase inhibitors for treating chronic diabetic complications, we will develop a new high-throughput model screening approach for selecting the convenient and reliable candidate of ARIs.