Bioinformatics analysis,expression,purification and activity detection of human R-spondin 2 protein
Objective To construct eukaryotic expression vector of human R-spondin 2(roof plate-specific spondin 2,RSPO2),express and purify fusion protein RSPO2-Fu-Fc,and detect the biological activity of RSPO2-Fu after resecting the Fc domain with 3C enzyme.Methods Based on bioinformatics method,the physical and chemical properties and structure of human RSPO2 protein were analyzed,and the truncated protein RSPO2-Fu which could exist stably in solution was screened.The gene encoding RSPO2-Fu protein was subcloned into pCMV-Fc vector,and then the plasmid pCMV-RSPO2-Fu-Fc was transiently transfected into HEK293F cells and expressed for 72 h.The fusion protein RSPO2-Fu-Fc was purified by Protein A chromatography column,the Fc domain was removed by 3C enzyme,and the RSPO2-Fu protein was further purified by using molecular sieve.Finally,the biological activity of RSPO2-Fu protein was detected by M50 Super 8 × TOPFlash.Results Bioinformatics analysis showed that RSPO2 was a hydrophilic unstable protein with 243 amino acids,a relative molecular mass of 28 300 and an isoelectric point of 9.42.The stability of RSPO2-Fu truncated protein in solution was greatly improved.The recombinant expression plasmid pCMV-RSPO2-Fu-Fc was constructed correctly as identified by colony PCR and sequencing,and RSPO2-Fu protein with a purity of 95％was obtained.TOPFlash results showed that the EC50 value of RSPO2-Fu was 1.5 × 10-12 mol/L.Conclusion After proper truncation of RSPO2 protein,stable expression of RSPO2-Fu protein can be obtained,which can obviously activate Wnt signaling pathway,and provides a better reference for RSPO2 related biological research in Wnt signaling pathway.
Mammalian protein expression systemRecombinant human R-spondin 2 proteinLuciferase reporter geneWnt signaling pathway
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