| Protein purification technology is widely used in biology,medicine,food and other fields.Among them,affinity chromatography is a very effective and simple method to purify a wide range of proteins.There are several advantages of affinity chromatography:strong specificity,high purity,and excellent final yields.But it also has certain disadvantages,such as requirement of fuse an affinity tag to the target protein.Affinity tags provide specific recognition capabilities,but also have a certain impact on the application of target proteins.The use of inteins as affinity tags reduces such expends.The split intein is spatially divided into N-terminal(I_N)and C-terminal(I_C).The I_N and I_Cof the split intein are used as the purification ligand and affinity tag,and the affinity tag can be cleaved at the same time of purification.Intein affinity purification system simplifies the purification process and provides new ideas for protein purification.In this study,an affinity purification system was constructed using Consensus fast Dna E(Cfa Dna E)split inteins.Cfa Dna E is an artificially constructed intein with the advantages of rapid splicing,high expression,and good stability.During the research,it was found that since the cleavage of the intein requires the assistance of the extein,it is necessary to insert the CFN tripeptide at the N-terminus of the extein,which affects the practicability of the affinity purification system.In this paper,molecular modification of Cfa Dna E intein was carried out to reduce the dependence of intein C-terminal cleavage reaction on extein.The specific research contents of this study are as follows:(1)Molecular modification of Cfa Dna E intein.Molecular modification was carried out using p ET28a-I_C-GFP preserved in our laboratory as a template.Mutation of EKD at positions121-123 of Cfa Dna E intein,ie I_C 20-22,to GEP,in order to reduce the dependence of intein C-terminal cleavage on the amino acids of+2 and+3 of the extein.(2)To study the dependence of the intein cleavage reaction on the+2,+3 positions of the extein.Tripeptide with CGG is inserted between the I_Cand the N-terminal of the extein.Cleavage reactions were performed on I_CG-C-GFP.It was found that when Glys were used as extein+2 and+3 positions,the intein could undergo a cleavage reaction with lower cleavage rate,which proved the dependence on the+2 and+3 positions of the extein of the modified intein declined.(3)Study the Selectivity of the intein cleavage reaction at the+1 position of the extein.According to theoretical analysis,it is believed that the C-terminal cleavage reaction of intein should be less dependent on the+1 position of extein.Tripeptide with Ser/Thr/Tyr/Gly/Met/Trp/Gln/Asn and the two Glys at positions+2 and+3 is inserted between the I_C and the N-terminal of the extein.The modified protein is subjected to a cleavage reaction.It was found that the fragmentation rate of the intein was also different when different amino acids acted as the first position of the extein.Among them,Asn cannot cleave the intein.Our analysis is that Asn competes with the Asn at the end of the intein,which interferes with the formation of the normal conformation of the intein.(4)Optimize the modified intein cleavage reaction conditions.Using I_CG-C-GFP as the experimental object,the p H and temperature conditions of the intein cleavage reaction were optimized.The experimental results showed that the optimum p H for the intein cleavage reaction was 7.0,and the optimum temperature was 37℃.(5)Application of the modified intein affinity purification system.Using the modified intein,the purification experiment was carried out with the first 3 positions of the extein were CGG.The GFP with a purity of 95.68%was successfully purified,the purification multiple was4.14,and the recovery was 29.50%.Compared with the unmodified intein affinity purification system,the purification effect is similar.The engineered intein was unaffected by extein alterations on small scale purification. |
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