Comparative Study Of The Insoluble Ulp1 Protease As Carrier-free Immobilizate And The Soluble Cellulose-binding Module Tagged Ulp1 For Oriented Immobilization

Small ubiquitin related modifier ubiquitin(SUMO3)is widely used as fusion partner to high level soluble production of target protein in E.coli.SUMO protease Ulp1 removes the SUMO tag from the fusion protein with its SUMO spatial structure dependence.Cleavage activity of the recombinant Ulp1 expressed by E.coli is much higher than other sequence-specific proteases been reported,and the low production yields make it a good choice to increase reuse efficiency of Ulp1 through immobilization.In this study,we fuse the GFIL8 hydrophobic peptide at the N-term of the Ulp1 to highly product carrier-free immobilizates in the form of active inclusion bodies by E.coli.;After cellulose binding protein(CBM)been fused at the N-term of the Ulp1,the fusion can be oriented immobilized with regenerated amorphous cellulose(RAC).After comparing the properties of the two immobilized enzymes,we found the following results:Comparing with the immobilized construct,the insoluble Ulp1 showed higher resistance to trypsin digestion,lower leaky amount at different storage temperatures.After it cleaved the protein substrate once,the aggregated Ulp1 lost its activity.The aggregates of freshly prepared Ulp1 and used for one time showed similar resistance to protease-K digestion,and binding capacity of thioflavin T and Congo red.Crosslinking of inclusion bodies by glutaraldehyde inactivated the insoluble Ulp1.Due to different advantages,the Ulp1 constructs as carrier-free and carrier-dependent immobilizates are hopefully used for SUMO tag-removal under different conditions and rapid separation from the reaction mixture.