Analysis of GPC3 protein structure and its expression purification
Objective To explore the structure of GPC3 protein and construct a prokaryotic sys-tem for its efficiently expression and purification.Methods Gene sequences were obtained from GenBank,and the structure of GPC3 protein(25-554 aa)was analyzed using online software Protparam,SOPMA and SWISS-MODEL.Select relatively advantageous combinations from different vectors and strains,optimize its expression conditions,and induce the expression of TrxA-GPC3 fusion protein.Separate and purify GPC3 pro-tein without TrxA label through dissolution and renaturation of inclusion body,affinity chromatography,label cleavage and molecular sieve purification.Results GPC3 is an unstable hydrophilic protein with the molecu-lar weight about 60.26 kDa,theoretical pI of 5.76,instability index of 41.27,Aliphatic index of 83.66 and grand average hydrophilicity of-0.312.It mainly consists of alpha-helices and random coil structures.The TrxA-GPC3 fusion protein with an introduced tag is more stable than the GPC3 protein alone.By comparison,the BL21(DE3)-pET32a-GPC3 engineered bacteria,which exhibit higher expression efficiency,were select-ed.Under the conditions of 0.5%glucose concentration and 0.1 mmol/L IPTG concentration,combined with an induction temperature of 20℃and an induction time of 16 hours,the protein expression level can be signifi-cantly increased.Through affinity chromatography,TrxA-GPC3 fusion protein with a purity of over 90%was obtained.The TrxA tag was then removed using thrombin,and finally,a single GPC3 protein was obtained through molecular sieve purification.Conclusions In this study,we analyzed the physicochemical properties and structure of GPC3 protein using bioinformatics,and successfully expressed and purified GPC3 protein in prokaryotic systems.This laid a foundation for the preparation or screening of highly specific antibodies and structural-functional studies,and provided scientific guidance for the expression and purification of other pro-teins.
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