Study On Construction And Properties Of C-terminal Cleavage System Mediated By Split Npu DnaE Intein

Affinity tags are widely used for the separation and purification of recombinant proteins,but it needs the additional time and steps to remove them,which greatly limits its application on a large-scale industrial processes.The intein mediated protein purification system can avoid the use of affinity tags in the purification of proteins,which has been widely studied in recent years.A new type of protein purification system mediated by Npu Dna E split intein was designed by using the specific recognition and high protein splicing ability between the N fragment?I_N?and C fragment?I_C?.The mainly studies were carried out as follows:?1?In order to make the I_N affinity ligand fragments correctly coupled to the epoxy activated Purose 6 FF,all the Cys in the I_N fragment were mutated by site directed mutagenesis.In addition,the histidine tag and Cys residue were added to the C terminal of I_N fragment for simplified the process of protein purification and chromatography coupling.What's more,the N137A and C+1A mutations were introduced into the I_C-GFP fragment to inhibit the splicing activity of the Npu Dna E intein.Finally,the recombinant strains of I_N affinity fragment and I_C fusion protein fragment were successfully constructed.Protein expression and affinity purification of recombinant bacteria were carried out,and the target protein with purity above 80%was obtained.?2?In order to study the C-terminal cleavage efficiency between the I_N and I_C-GFP fragments,the factors(p H,temperature,DTT and Zn²⁺concentration)effecting the cleavage efficiency were optimized.After the mutation,the I_N fragment was almost unable to form intramolecular disulfide bonds and could achieve rapid C-terminal cleavage under non-reducing conditions.?3?The relationship between the different concentration of I_N fragments and the density of ligand were studied to improve the ligand density of I_N media,and the optimal concentration of I_N was determined.In this thesis,the static adsorption and dynamic adsorption conditions of I_N medium were investigated.The static adsorption results showed that the saturated adsorption capacity of the medium reaches the maximum under the condition of p H 7,while the adsorption capacity of the medium is lower under the condition of p H 6.The dynamic adsorption capacity of I_N medium was measured by the 10%protein breakthrough curve.The experimental results showed that the dynamic adsorption capacity of I_N medium was 9 mg·m L^-1 wet resin under the condition of p H 7.After 6 mol·L^-1 guanidine hydrochloride was used for media regeneration,the dynamic adsorption capacity of the medium was maintained at 90%after repeated use of 5 times.?4?GFP was used as a model protein to investigate the purification process of I_N medium.The protein was purified by 1 m L prepacked column using?KTA purifier chromatography system.The recovery rate and purification factor of the target protein are about 39%and 12.1,respectively,and the purity of the protein is about 97%.I_N affinity chromatography medium has good stability and great application value.