Establishment And Application Of PAPS Purification And Detection System Based On PAPS Binding Protein HAL2

Sulfur,one of the important non-metal elements,is indispensable for life activities.After sulfate enters into cells,it can be activated by ATP sulfurylase to form adenosine-5'-phosphosulfate?APS?,which could be reduced to sulfite by APS reductase.Alternatively,APS could be further phosphorylated by APS kinase to form3'-phosphoadenosine 5'-phosphosulfate?PAPS?,the sole sulfate donor for intracellular sulfation.PAPS involves the synthesis of glucosinolates and heparin-based polysaccharides.Sulfotransferase catalyzes sulfation of different compounds with PAPS as a substrate,and the by-product is 3',5'-bisphosphate adenosine?PAP?.3'nucleotidase catalyzes dephosphorylation of 3'phosphate from PAP to form AMP,and from PAPS to form APS.PAPS content is very low in biomaterials.Commercial PAPS is expensive with low purity.In this research,we used the PAP/PAPS binding protein to establish a method for purification and enrichment of PAP/PAPS from biological materials.The 3',5'-bisphosphate nucleotidase homolog of yeast?HAL2?hydrolyzes PAP or PAPS to produce AMP or APS,respectively.Mg²⁺is required for HAL2 binding and hydrolysis of PAP/PAPS,and the presence of Ca²⁺keeps the binding of HAL2and PAP/PAPS,but eliminates the hydrolysis activity.In this research,the fusion protein MBP-HAL2 of maltose binding protein?MBP?and HAL2 was obtained by gene cloning,protein expression and purification,andit was used for enrichment and quantitative analysis of PAP/PAPS.The purified MBP-HAL2 protein is immobilized on MBP affinity column,and will bind the PAP/PAPS in the sample column can be obtained by eluting the column with EGTA which chelates Ca²⁺,and while the sample runs through column in the presence of Ca²⁺,PAP/PAPS on the column can be obtained by eluting the column with EGTA which chelates Ca²⁺,and quantified by HPLC.According this method,PAP and PAPS contents in E.coli were determined to be 1.076±0.007 and 0.997±0.009 mg/g wet pellet,respectively.PUF?Pumilio and FBF?is an RNA-binding protein that exists in eukaryotes and mainly participates in post-transcriptional regulation of genes by specific binding to the 3'-UTR of target gene mRNA,and involves in embryogenesis,cell development and differentiation.The corresponding codes of the RNA binding domain?RBD?of the PUF protein and its paired nucleotides had been resolved,and it had been shown that RBD of PUF protein forms a rigid RNA-binding backbone.Previous results indicate that binding of the RNA binding backbone to the coding region of the target RNA will result in gene down regulation.We proposed to use this strategy to reduce the hydrolysis of PAP/PAPS and increase the intracellular PAP/PAPS content of E.coli by reducing the expression of 3',5'bisphosphate nucleotidase,CysQ.Based on the quantitative PCR results,we found that the attempt to reduce CysQ expression by this method was failed.The reasons might lie in that the intracellular content of CysQ mRNA is too low,or the designed PUF can not bind the target effectly to downregulate gene expression.Further studies need to be carried out for elucidating this question.In summary,we established a PAP/PAPS enrichment and analysis method based on MBP-HAL2 fusion protein,and successfully applied to analyze PAP/PAPS in E.coli.