The Study Of The Immobilization Of Ulp1 Protease And Its Applications

Small ubiquitin-related modifier(SUMO)fusion expression system is widely used to express easy misfolding,unsoluble and toxic proteins.6*His tag is mostly used to purify proteins in many laboratories,but it is easy be wrapped into target proteins for its small molecular weight.SUMO tag can be recombined behind the 6*His tag to avoid wrapping 6*His tag and increase the binding efficiency with Ni-NTA.Conversely,some small molecule proteins may damage their functions due to introduction of the big molecule,SUMO tag.Ubl-specific protease(Ulp1)can perfectly solve this problem.It can cleave the epsilon-linked peptide bond between the C-terminal glycine of the mature SUMO and the lysine epsilon-amino group of the target protein by identify tertiary structure of SUMO.It has no effect on the further study of the target proteins as it cleave off the SUMO by identify its tertiary structure and don't keep any amino acid residues.Ulp1 no matter is commercial or purified in the lab is expressed in soluble form which is easy to use and widely used for protein purification.However,there are some problems for the applications of Ulp1,the activity of Ulp1 will gradually lost during preservation especially after long time storage,even at-80?.Free Ulp1 can't be reused,and further purification precession is needed to remove Ulp1 and SUMO from cleaved proteins.we are targeting to fabricate an active and stable enzyme through covalent immobilization.The results show as followings:1.Considering the information that H514 and C580 residues,but not any lysine residues are important activity sites for Ulp1,immobilization strategies for protein coupling through the ?-amino groups of lysine residues were employed.Therefore,we synthesize CNBr-activated Sepharose,CHO-Agarose and NHS-activated Sepharose.The binding efficiency of these beads were tested and shows good binding effenciency.2.It is useful to immobilize proteins on these such beads,so we used CNBractivated Sepharose,CHO-Agarose and NHS-activated Sepharose for Ulp1 immobilization.NHS-activated Sepharose was shown as the most efficiency matrix for immobilization with no effect on the cleavage activity and the binding capacity was estimated to be 1.68 mg/ml of the matrix.3.The enzymology properties of immobilited Ulp1 are studied,and results show that:Due to the insufficiently mixing with substrates,3 fold amount of immobilized Ulp1 as the soluble one was used and found the immobilized Ulp1 can get similar reaction velocity with free Ulp1.Both Ulp1 have a broad pH adaptation which can work well within pH 4.5-11.0.These two forms Ulp1 also can resist to some small molecules,such as 800 mM NaCl,1M urea,100 mM DTT and 400 mM imidazole but can't bear SDS.we tested the effect of organic reagents on the activity of Ulp1 and detected that there is no significant effect on enzymatic activity of the immobilized Ulp1 both 20 % ethanol and 15 % DMSO,but DMSO completely inhibit the activity of free Ulp1.4.The stability of immobilited Ulp1 are studied,and results show that:the free Ulp1 lost about 84 % of activity after 24 hours incubation at 37 ? and the immobilized one retained 96 % of activity even after 36 hours.Under 25 ? storage conditions,only 7 % decreasing of activity for the immobilized Ulp1.Immobilized Ulp1 can stored at 4 ? for more than 50 days.The immobilized Ulp1 retained full activity at pH 4.5-8.5,only a slightly activity decreasing at pH 10.5.It seems that Ulp1 is an acid-tolerant protease and immobilization enhances its pH stability.And immobilized Ulp1 shows reusability for more than 15 batch reactions.5.Fast purification strategy applied in no tag GR purification obtains pure aimed products within one hour.This way may be a feasible approach to replace the streamlined method in industrial to produce various no tag proteins at the same time.