Pparγ With Gst Fusion Expression And Be Fixed On The Surfase Of Micro Nano Magnetic Particles

Based on the target proteins immobilized on magnetic nano materialsurface, by magnetic separation and chromatographic analysis of rapiddetermination of unknown content in the mixture of candidate ligand affinityscreening of combinatorial libraries of new technology, can effective lowcost high throughput for rapid screening for target proteins of high affinityligand. In this paper,using prokaryotic expression vector pGST-MOLUCsystem encoding a molecular mass of approximately26KD Schistosomajaponicum GST expression fusion tag, followed by multiple cloningsites.Insert into target gene fragment of PPAR gamma ligand bindingdomain (LBD) after cloning sites, to make the the fusion protein and GSThave the same open reading frame, further purification to obtain the targetproteins. Attempting through the human source PPAR gamma model toexplore the beads immobilized target proteins, establishment of magneticseparation screening technology, which can rapid screen of high affinityligands for PPAR gamma.1. Recombinant expression of GST tag fused protein PPARγ-LBDFirst of all, gene cloning PPARγ-LBD construction of recombinant vectors GST-hPPARγ-LBD, which was transferred into Escherichia coli toinduce expression.Upon purification, the soluble protein purity are obtainedabout90%,which was detected binding with affinity ligand rosiglitazone,shown to the specific binding percentage of66.12%, having biologicalactivity, which can be used for the follow-up PPARγ function activityresarches. And this will be a foundation for the establishment of magneticseparation technology for screening its high affinity ligand.2. Magnetic beads immobilized target protein, investigating theretention activity of target proteinsIn this paper，immobilized not fusion target proteins GST tag on thesurface of beads and tested its retaining activity. Attempting aminoimmobilization and thiol site immobilization. GST tag was modificated byfree reagent SS-mPEG and N-ethyl maleimide, both GST residual activitywere above60%, but when fixed in hydrophilic beads surface, the retentionactivity were between1%and5%, which can not meet the requirementsand immobilized technology remains to be optimized.3. Exploration screening method for high affinity ligandsFirst of all, using GST activity determination methods to measured thetwo substrates GSH and CDNB of Km, GST for GSH Kmwas0.11mmol/L,for CDNB was0.69mmol/L. GST reaction in the presence of significantproduct inhibition, fixed GSH concentration, GS-DNB against GST forcompetitive inhibition Kiwas (4.8+0.4) mmol/L (n=2), fixed CDNB concentration GS-DNB against GST noncompetitive inhibition of Kiwas(34±3) mmol/L(n=2). GS-DNB on the tag GST inhibition effect of liveralkaline and acidic GST isozymes are differed significantly (P<0.05).Andthen using enzyme kinetics of initial velocity method for the determinationof dissociation constants about a series of butanediamine derivatives.N,N’-bis-ethacrynyl-1,4-butyldiamine had the highest affinity for GST inthese inhibitors, which was the derivative of ethacrynic acid connected withbutanediamine. With inhibitory potency in nmol/L, compared to ethacrynicacid not connected with dimethyl amine, raising nearly threemagnitudes.The secondly strong inhibitor wasN,N’-bis-(4-(n-butyl)-benzoyl)-1,4-butyldiamine, which was the derivativeof ethacrynic acid connected wuth butanediamine.Based on the measured inhibition constants of GST inhibitors/ligands,high affinity ligands can be selected and and transferred as affinity labelingreagents for GST; magnetic beads immobilized with such affinity labellingreagents can be used for GST site selectivity attachment, thereby tag fusedtarget protein can be immobilized with remained activity for the screeningof target protein mixtures ligand libraries.