Study On The Performance Optimization Of The C-terminal Cleavage System Mediated By Split Intein

Affinity chromatography technology is one of the effective methods for the separation and purification of recombinant proteins.It has the characteristics of easy operation and high purification efficiency.However,affinity chromatography technology usually requires the introduction of special affinity tags into the target protein.There are endonuclease method,chemical method,etc.commonly used in industry to remove the label,but the processing method is often time-consuming,labor-intensive and very expensive.As a special protein,intein contains two discrete protein fragments,I_N and I_C.Through a series of self-catalyzed reaction processes such as rearrangement,transesterification,and cyclization,the intein can be excised from the precursor protein and the protein polypeptide chains(Extein)at both ends are connected by a natural peptide bond.That is to realize the rearrangement of protein structure through protein self-splicing.Based on this,the I_N and I_C fragments are designed as affinity ligands and tags,respectively,and the protein purification is completed by the affinity of the two,and the tags are removed by the self-cleavage of the intein,effectively avoiding tag pairs.The influence of protein simplifies the operation of protein purification.In the actual application process,it was found that the affinity chromatography medium prepared based on Cfa Dna E intein can obtain higher purity for protein purification,but the new medium has a lower regeneration rate and poor reusability.This study included Peptide-containing as the research object,guided by the results of molecular docking,combined with protein engineering,the Cfa Dna E intein was modified to obtain the optimal regeneration conditions of the medium.In this study,a new I_C protein was successfully constructed,which effectively improved the elution effect without significant influence on the recognition connection and medium loading.The specific research content is as follows:(1)Study the protein structure of Cfa Dna E intein.It is necessary to establish an I_N protein model based on the base sequence of I_N,perform molecular dynamics simulation,analyze the flexible region in the I_N protein according to the RMSF value,and combine protein engineering to modify part of the I_N protein.(2)Predict the impact of mutations on the stability of I_N protein.The model was mutated according to the I_N protein structure,and the I_N protein model with single mutation,double mutation and triple mutation was obtained.Perform molecular dynamics simulation on the mutant protein model,analyze the Rg value changes of different mutant proteins,and obtain the optimal mutant protein.(3)Verify the effect of mutation on the stability of I_N protein and the ability to recognize and connect.According to the results of molecular simulation,the I_N protein was modified by PCR technology,and the optimal I_N protein was obtained,and the fragmentation reaction was carried out to analyze the influence of the mutation on the stability of the I_N protein and the ability to recognize and connect.(4)To study the interaction between Cfa Dna E intein I_N and I_C.In this study,a three-dimensional model of I_N and I_C protein was constructed based on the amino acid sequence of I_N and I_C fragments,and the molecular docking of I_N and I_C was carried out on the basis of the model,combined with the existing three-dimensional models of intein such as Npu Dna E,Ssp Dna E,etc.Score with the docking model and select the best protein complex model.Taking this model as the research object,analyzing the size of the interaction force and the area of action,the results show that there are two hydrogen bonds at the 12th,14th,and 15th amino acid positions of the I_C.Therefore,it is considered that the 12-15th amino acids are mutual Role of key areas.(5)Construction of I_C mutant protein.In order to further optimize the regeneration conditions of the affinity chromatography system,the main research method of this experiment is to combine protein engineering.It is planned to insert glycine into the I_C to reduce the stability.In order to study the influence of the number of glycine insertions on the stability of I_C,I_C-3G,I_C-6G,I_C-12G,I_C-18G were inserted between positions 12 and 15 of the I_C protein.(6)Study the effect of mutation on I_C protein.After inserting 3 or 6 glycines in the upper column elution experiment,the elution effect of I_C-GFP-D protein under 0.1 M Na OH elution conditions is obvious.By analyzing the effect of mutation on the recognition of the connection through the cleavage reaction,it is found that the insertion of glycine will cause the cleavage rate to slight decrease,but the cleavage rate has no obvious effect.At the same time,in the static adsorption experiment,inserting glycine will decrease the loading,but it is still within the acceptable range.The mutant protein was applied to protein purification,and it was found that it did not significantly affect the purification effect.In repeated loading experiments,I_C-6G can maintain a regeneration rate of about 95%,which is significantly higher than that of I_C.In summary,inserting six glycines is the optimal mutant protein.