Study On The Relationship Between Non-Natural Extein Sequences And Fracture Properties In Cfa DnaE Intein

Inteins can release themselves from immature precursor proteins through a series of reactions during protein maturation.Based on this theory,the N-terminal fragment(I_N)of the broken peptide-containing peptide is designed to bind to the affinity ligand and chromatographic medium,and the C-terminal fragment(I_C)is designed to be fused and expressed by the tag and the target protein,and the target protein is released through the self-shearing reaction of the peptide-containing protein after specific recognition.Our laboratory prepared affinity chromatography media to purify proteins using peptides contained in Cfa DnaE.However,the completion of protein splicing with peptides requires exteins,that is,the first three amino acids of the target protein are Cys,Phe,Asn,which will make the target protein introduce exogenous amino acids while completing purification,and the introduction of exogenous amino acids may affect the properties of the target protein.In order to solve this problem and expand the application scope of the purification system,this paper carried out molecular modification of the peptide contained in Cfa DnaE,and used green fluorescent protein(GFP)as the model protein to explore the effect of the target protein on the splicing reaction of the containing peptide when the first three amino acids are unnatural amino acid sequences other than Cys,Phe and Asn.The specific research content is as follows:(1)The original Cys,Phe,and Asn residues between the I_C fragment and GFP protein were mutated,and five non-natural amino acid sequences were selected to verify the dependence of the original intrinsic peptide shear reaction on the first three amino acids of the target protein,and none of the results detected the production of the shear reaction product GFP indicating that none of the intrinsic peptide shear reactions occurred,indicating that the original intrinsic peptide shear reaction is dependent on the first three amino acids of the target protein amino acids of the target protein.(2)The dependence of the modified intrinsic peptide splicing response on the+2 and+1positions of the target protein was investigated after mutating Glu,Lys,and Asp,which are located near the critical shear site in I_C,to Gly,Glu,and Pro,respectively.Eight amino acids,Asp,Phe,Tyr,Asn,Arg,Trp,Ala,and Gly,were selected to investigate the+2 position.Three amino acids,Ser,Thr,and Met,were selected when studying the+1 position.The results showed that both+2 and+1position breakage reactions of different amino acids as target proteins could occur,only the rate of breakage reaction occurrence and final breakage rate differed.The final breakage rate of hydrophobic amino acids as the+2 position was more than 80%,and the lowest breakage rate of hydrophilic amino acids as the+2 position was about 75%.This indicates that the dependence of the+2 position of the modified intrinsic peptide on the target protein was reduced.The conventional three amino acids with nucleophilic group as the+1 position all had more than 80%breakage rate,and the breakage reaction occurred more completely compared with the other amino acids as the+1 position.It indicates that the+2 position and+1 dependence of the modified intrinsic peptide on the target protein is reduced.(3)To study the combined selectivity of the modified intrinsic peptide reaction on the+1 and+2amino acids of the target protein.Eight mutants were selected to study the combined effect of the first two amino acids of the target protein on the splicing reaction.The final experimental results showed that all eight mutants and the I_N cleavage reaction could occur and the final cleavage rate was above70%.This indicates that the dependence of the modified intrinsic peptide on the first three amino acids of the target protein is reduced.(4)To further verify the applicability of the mutated intrinsic peptide purification system,GFP and ethanol dehydrogenase(ADH)and Ic were selected for fusion expression,and the amino acid sequences of the two proteins were combined to make the first amino acid of the target protein Ser by PCR.After constructing the chromatography medium and using it to purify the two proteins,the purity of both proteins was above 90%.