Prokaryotic Expression,Purification And Enzyme Activity Determination Of OsSOT1

Sulphotransferase?SOTs?is a family of highly conserved proteins that catalyze the transfer of sulfo(SO^3-)to the amino or hydroxyl groups in the substrate and have important physiological functions in the cell.At present,SOTs are generally expressed by prokaryotic expression and purified by affinity chromatography.The kinetic constants are determined by isotope method,absorbance method,etc.The enzyme biological data is combined with physiological data to analyze its specific biological functions in vivo.However,there are some shortcomings and problems to be solved in the system,such as low concentration and purity of purified protein,PAPS as a general substrate is expensive and easy to decompose,the enzymatic determination operation is complicated and time-consuming.This study initially explored ways to solve these problems.Using the homologous alignment of the known gene AtSOT12?At2g03760?in the Oryza sativa Indica Group gene pool,the most homologous OsSOT was obtained,which was shown to be OsSOT1 by comparison with the reported sequence.According to the latest nomenclature,OsSOT1 and AtSOT12 can be classified into one family,suggesting similar structural and functional similarities.At present,some enzymology information of Arabidopsis thaliana and human SOTs has been reported,but the analysis of rice SOTs only stays on the genetic and physiological information.No related enzymology information has been reported.This will lead to the following aspects.Exploring different labeling and expression conditions to solve the problem of easy formation of inclusion bodies in the expression of AtSOT12 and OsSOT1,the results showed that increasing the NaCl concentration to 0.2-0.4 M in LB medium can reduce the formation of inclusion bodies;MBP as a fusion label can be significant Increased solubility of the fusion protein,but not conducive to label removal;6His and GST double label can be used to purify higher purity protein samples.The 3'-Adenosine-5'-phosphate sulfate?PAPS?was synthesized by a multi-enzyme reaction system,and the optimal synthesis conditions were discussed.The results show that the most suitable conditions are pH 8-8.5,temperature 35?,PK and PEP can increase the synthesis rate of PAPS?>90%?;Using a strong anion exchange column,a KCl solution?pH 4.5?was selected for gradient elution?200-500mM?to obtain a high concentration,high purity PAPS,but there may be degradation of PAPS during the purification process.Based on the property of the Hal2 mutant G236V,which has a PAP affinity of5592 times that of PAPS,SOT detection system coupled with G236V/MK/PK/LDH enzyme was established.Some substrates of AtSOT12 were determined by this system.Compared with related reports,the measured K_m values are similar but kcat is larger.The possible reason may be the effect of solvent ethanol on the established system,or the reaction system added with glycerol?Since the substrate is fat-soluble,the addition of glycerol may promote the reaction?,and a sufficient amount of PAPS is added,and the protein purity is high.Using this system to explore some substrates of OsSOT1,the results showed that OsSOT1 can catalyze certain phytohormones such as salicylic acid?K_m=360.94?M?and 24-epibrassinolide?K_m=5.83?M?,which can also catalyze 5-Hydroxyphthalic acid,pregnenolone and dehydroepiandrosterone.In summary,the subject systematically constructed a system for detecting the enzymatic information of SOT,and measured some substrate enzyme information of OsSOT1,and established a high-throughput,convenient SOTs activity detection system.The research on rice and other specific SOT has certain basic significance.