Eukaryotic Expression And Purification Of Omentin-1 Protein

Background: Omentin-1 is a novel adipokine,mainly expressed by mesenchymal vascular cells of visceral adipose tissue.Omentin-1 has also been known as the intelectin-1(ITLN1),intestinal lactoferrin receptor,endothelial lectin HL-1 or galactofuranose-binding lectin.Studies have found that Omentin-1 is involved in physiological and pathological processes such as maintenancing energy homeostasis of body,glucose metabolism,cardiovascular protection,antiinflammation,antioxidative stress and nerve cell function regulation,and closely related to the occurrence and development of obesity,diabetes,cardiovascular diseases,tumor and cerebral infarction.Recombinant Omentin-1 protein has been widely used as an important tool for Omentin-1 function verification and exploration of its mechanism actions in related diseases.Since the mature Omentin-1 protein in plasma is in a trimer state,and the commercial Omentin-1 protein is mostly expressed by iprokaryotic,the protein structure cannot satisfy this characteristic.In order to maximize the biological activity of plasma Omentin-1 protein and further analyze the physiological and pathological function of Omentin-1 protein,it is important to obtain Omentin-1 trimeric protein.Objective: Establish the eukaryotic expression system of Omentin-1 protein,purify the Omentin-1 protein expressed by eukaryotic expression system,and identify the relative molecular mass,concentration and purity of purified Omentin-1 protein.Methods: Based on the vector pEGFP-N1(4733bp),two plasmids,Flag-ITLN1(4905bp)and ITLN1-Flag(4896bp),with similar molecular masses were constructed.Double digestion experiment and DNA sequencing were used to identity plasmid.Optimized transfection conditions for plasmids transfection with fluorescence intensity of pEGFP-N1 plasmid.Flag-ITLN1(4905bp)and ITLN1-Flag(4896bp)were transfected into HEK 293 cells with the optimized transfection protocol,and pEGFP-N1 plasmid transfection wells were set to evaluate transfection efficiency,then establish the eukaryotic expression system of Omentin-1.Quantitative reverse transcription-PCR was used to detect the difference of Omentin-1 gene expression after plasmid transfection(24h).Western blotting(WB)was used to detect the differentce of the expression of Omentin-1 protein in cell culture medium after plasmid transfection(24h),and the system of omentin-1 protein eukaryotic expression with relatively high expression of Omentin-1 protein was screened.The cell culture medium of the established eukaryotic expression system of Omentin-1 protein was collected.Purification of Omentin-1 protein in cell culture medium by diphenylsulfone resin coupling method.Ultrafiltration with molecular weight cutoff of 30 kDa and 100 kDa were used respectively to isolate the mixed protein,the relative moleclar mass and purity of purified Omentin-1 protein were identified by SDS-PAGE gel electrophoresis,the nature identified by WB and mass spectrometry,and the concentration was determined by BCA method.Results:1.The expression of Omentin-1 gene of HEK 293 cells and protein in the culture medium were higher than that of ITLN1-Flag plasmid after transfection.2.The eukaryotic expression system of Omentin-1 with Flag-ITLN1 plasmid transiently transfected HEK 293 cells was established successfully.3.The Omentin-1 protein in cell culture medium was purified successfully by diphenylsulfone resin coupling method.4.The molecular mass of purified Omentin-1 protein was about 130 kDa,the purition over 90%,and the concentration was 0.035mg/mL.Conclusions:1.The Omentin-1 eukaryotic expression system based on transiently transfecting HEK 293 cells with Flag-ITLN1 plasmid expresses recombinant Omentin-1 protein.2.Recombinant Omentin-1 protein expressed by eukaryotic expression system is a trimer.