Preparation of recombinant human Siglec-1 protein and screening of natural ligands based on affinity ultrafiltration-liquid mass spectrometry
Objective To construct a prokaryotic expression vector of sialoadhesin(Siglec-1),express and purify the recombinant Siglec-1 protein,and construct an ultra-filtration affinity-liquid chromatography-mass spectrometry(UF-LC-MS)technique to screen natural ligands of Siglec-1 protein.Methods The prokaryotic expression vector of Siglec-1 recombinant protein was constructed by DNA recombination technology.The Siglec-1 recombinant protein was expressed and purified,the purity of Siglec-l recombinant protein was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE),and the protein was purified by Ni column affinity chromatography.An UF-LC-MS technique was established to screen the affinity of six main components(chlorogenic acid,2-rosmarinic acid,3-glycyrrhizic acid,4-chicoric acid,5-glycyrrhetinic acid,and 6-ferulic acid)in Lonicera japonica,Glycyrrhiza uralensis,Rosmarinus officinalis and Cichorium intybus to Siglec-1 protein.Calculate the specific binding rates of each analyte by comparing the peak areas of the analyte in the ultrafiltration solution of the sample group and the protein denaturation group.Results The double-enzyme digestion and sequencing identification proved that the recombinant expression plasmid pET-22b-Siglec-1-His was constructed correctly,the relative purity of purified human Siglec-1 recombinant protein was over 90％,the UF-LC-MS screening system was established,and glycyrrhetinic acid was screened out.Glycyrrhetinic acid can specifically bind to Siglec-1 protein.Conclusion A high-purity and recombinant human Siglec-1 protein with certain chemotactic activity was successfully expressed.It was screened out that glycyrrhetinic acid can specifically bind to Siglec-1 protein.
Siglec-1recombinant proteinaffinity ultrafiltration-liquid mass spectrometryglycyrrhetinic acidnatural ligands
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