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

Protein purification technology is widely used in medicine,food,chemical engineering and other fields,and affinity chromatography is an effective method.It is easy operation and high purification efficiency.However,affinity tags need to be introduced into the target protein in affinity chromatography.In general,these tags will interfere the function of the protein and need to be removed by protease,which is time-consuming and expensive.The split intein contains two spatially discontinuous fragments,N fragments?I_N?and C fragments?I_C?.The I_N and I_C fragments can be used as affinity ligands and tags,respectively.The target protein can be purified by affinity adsorption.It can be used in the constructed affinity purification system without additional tag removal,which effectively avoids the use of protease and simplified protein purification.Therefore,it is a better choice to apply the split intein to protein purification.Consensus fast DnaE?Cfa DnaE?intein is a novel artificially split intein.It has high splicing activity and high environmental tolerance.However,it has not been applied in the field of protein purification.Therefore,in this study,a novel affinity purification system based on Cfa DnaE intein was constructed by modification of the intein.The specific research contents of this study are as follows:?1?In order to establish a novel affinity purification system based on Cfa DnaE intein,this study mutated all cysteine to alanine by molecular means in the I_N fragment of Cfa DnaE,the C-terminus of I_N introduces a His tag and a cysteine for the purification of the I_N fragment and the vector coupling,respectively.The I_C fragment and the target protein GFP were fused for purification of the protein.On the basis of I_C-GFP,two key amino acids,Asp136 and Cys+1,were deleted,and the intein fragment cleavage reaction was prevented,which was used to determine the chromatographic properties of the medium.Three engineering strains of E.coli BL21?DE3?/pET30b-I_N,E.coli BL21?DE3?/pTXB1-I_C-GFP and E.coli BL21?DE3?/pTXB1-I_C-GFP-D were successfully constructed,and used in media preparation,protein purification and media performance determination.?2?In order to further optimize the expression and purification conditions of the purification system,the density of bacteria,inducer concentration,induction time and induction temperature of E.coli BL21?DE3?/pET30b-I_N and E.coli BL21?DE3?/pTXB1-I_C-GFP was optimized,respectively.The optimal expression condition of I_N protein was that when the OD₆₀₀ of the cells was 0.6,IPTG was added at a final concentration of 0.2mmol?L^-1,and the expression was induced at 18°C for 15 h.The optimum expression condition for I_C-GFP protein was that when the OD600 of the cells was 0.6,IPTG was added at a final concentration of 0.4 mmol?L^-1,and the expression was induced at 18°C for 15 h.The optimal removal of impurities concentration and elution concentration of the I_N protein was150 and 500 mmol?L^-1,respectively.The optimal removal of impurities and elution concentrations of I_C-GFP fusion protein were 250 and 500 mmol?L^-1,respectively.?3?In order to further reveal the intein cleavage and inhibition cleavage conditions in the purification system,this study compares the cleavage rate of I_N and I_C-GFP proteins under different pH,temperature,DTT concentration and Zn²⁺concentration.The results showed that the Cfa DnaE intein cleavage rate did not change significantly between pH6-8.The cleavage effect was significantly different at 4°C-80°C.The best cleavage and inhibition cleavage temperatures were 37°C and 4°C,respectively.The rate of cleavage reaction was accelerated in the DTT concentration range of 5-50 mmol?L^-1,and at the DTT concentration of 5 mmol?L^-1,the cleavage rate was 90%after 6 h,and at the DTT concentration of 50 mmol?L^-1,the cleavage rate was 98%after 6 h.The cleavage rate was reduced at a concentration of 0.5-5mmol?L^-1 of Zn²⁺.Compared with the blank control,the cleavage rate was reduced by 31%at the 0.5 mmol?L^-1 Zn²⁺,and decreased by 47%at the 1 mmol?L^-1 Zn²⁺.Taking the cleavage rate as the index,the optimal cleavage conditions of the intein were pH7,37°C,DTT concentration of 50 mmol?L^-1;the best inhibition cleavage condition were pH6,4°C and Zn²⁺concentration of 1 mmol?L^-1.?4?A novel affinity purification system for Cfa DnaE intein was applied in GFP purification.In this study,the I_N ligand was covalently coupled with the activated agarose carrier to prepare a novel I_N affinity chromatography medium,and the optimal regeneration conditions were explored.The effect of the new medium on the purification of GFP was as follows:I_C-GFP-D had a saturated adsorption capacity of 66.48 mg?mL^-1,a dynamic loading of 30 mg?mL^-1,and the dynamic loading of the regenerated medium was 29.81 mg?mL^-1 under0.1 mol?mL^-1 NaOH.The purity of the GFP protein was 98.21%,and the recovery rate was29.33%.