Design,synthesis And Characterization Of Affinity Labeling Reagents For Gst Tag

The Schistosoma japonicum GST?sjGST,26KD?is a commonly used fusion protein tag.Site-selective immobilization of fusion proteins based on affinity labeling of fusion expression tag GST?sjGST?are an important strategy.GST is a homodimer with two active centers containing two hrdrophobilic binding sites.The labeling reagents capable of crosslinking two active centers can be used for immobilization of sjGST fused proteins on magnetic beads for screening of small molecule ligands through magnetic separation and liquid chromatography detectionBased on the distance between two active centers of the sjGST dimer and the position of cysteine residues near the active centers,two large benzene rings as the binding groups,bromoacetyl as the covalent group for sulfydryl,were conmbined to yield monovalent labeling reagent Br-I;and then a selected flexible chain linked with double Br-I to obtain a bivalent labeling reagent Br-II.The products under the action of sjGST in the presence of GSH have higher affinity and reversible labeling of the sjGST.Br-II for labeling sjGST,and immobilization of sjGST on magnetic beads were then characterized.After screen a number of synthetic routes,p-methoxyphenol was finally selected as the raw material,which was conjugated with p-acetylamino benzoic acid via N-ethyl glycine to obtain the monovalent labeling reagent?Br-I?for coupling with the amino magnetic beads.Double Br-I were linked by diethylenetriamine to get divalent labeling reagent?Br-II?for the coupling with the carboxyl magnetic beads.Structures were confirmed by ¹H-NMR,¹³C-NMR and HRMS.The labeling rate constant of Br-II at equival and 100-fold molar equivalents to sjGST were 0.00783 min^-1 and 0.236 min^-1.Apparent inhibition constant to sjGST was micromolar levels.The binding ratio of the Br-II to the active site of the GST was 1: 1.Apparent inhibition constant of product with GSH to the sjGST was micromolar levels.The GST dimer cross-linked by Br-II was unobserved by SDS-PAGE.The amino group was crosslinked with the carboxyl magnetic beads by the action of DCC.The beads can irreversible immobilize to sjGST,and reversible immobilize to sjGST in presence of GSH.The irreversible immobilization reached stable in 6 h and kept stable for further 6h,the immobilization capacity was about 24 ± 2 ?g/g.The reversible immobilization reached stable in 45 min and kept stable for further 1.5h,the immobilization capacity was about 26 ± 2 ?g/g.The non-specific binding of alkaline phosphatase was negligible.Nonspecific adsorption for small molecules was characterized with an hydrophobic fluorescent substrate.It was found that irreversible immobilization had some non-specific adsorption for the hydrophobic fluorescent substrate,but it can be reduced by further reaction with Br-I and GSH.This method should be applied to immobilization of sjGST via Br-I conjugated amino magnetic beads,and Br-I was easy to synthesis.Br-I was thus characterized further.The labeling rate constant of the Br-I at equival at equival and 100-fold molar equivalents to the sjGST were 0.01074 min^-1and 0.205 min^-1.Apparent inhibition constant of Br-I to the sjGST was micromolar levels.The binding ratio of the Br-I to the active site of the GST was 2: 1.Apparent inhibition constant of product of Br-I with GSH to the sjGST was micromolar levels.The carboxyl group of Br-I was crosslinked with the amino magnetic beads by the action of DCC,The beads can irreversible immobilized to sjGST,and reversible immobilized to sjGST in presence of GSH.The irreversible immobilization reached stable in 6 h and kept stable for further 6h,the immobilization capacity was about 25 ± 2 ?g/g.The reversible immobilization reached stable in 45 min and kept stable for further 1.5h.The immobilization capacity was about 28 ± 2 ?g/g.The non-specific binding for alkaline phosphatase was negligible.Nonspecific adsorption for small hydrophobic fluorescent substrates was observed,which was mitigated by further treatment with Br-I and GSH.In summary,Br-II and Br-I both upon conjugating with magnetc particles can be irreversible immobilize sjGST with the similar immobilization capacity.The irreversible immobilization needed further treatment with Br-I to reduce nonspecific binding.Synthesis of Br-I is simpler with a higher yield.So it is more advantagious to irreversible immobilize sjGST with Br-I.Reversible immobilize to sjGST with Br-II is also applicable.Therefore,these two strategies to immobilize sjGST are expected to be used for the site-spective immobilization of sjGST fusion proteins.