Intein-Mediated Soluble Expression And Purification Of Antibody Fragments And Immunotoxin In E.Coil

Background and aimImmunotoxins are recombinant proteins that combine antibodies with toxins,which are used to target and kill cancer cells.The fragment scds Fv is commonly used as the antibody part of immunotoxin.The peptide chain and disulfide bond exist simultaneously between the heavy chain and the light chain of scds Fv.There are different sources of toxins in the immunotoxin.The commonly used toxin is pseudomonas exotoxin A,an exotoxin secreted by pseudomonas aeruginosa.The modified fragment PE38 KDEL that comes from PE is the main form of current application.Inclusion bodies are very easy to form when recombinant immunotoxin is expressed in Ecoli.They need to go through complicated protein refolding process to recover protein activity,and the refolding efficiency is low,which increases the time cost and economic cost in the process of forming recombinant protein production.The soluble label can improve the soluble expression of immunotoxin,but the recombinant protein containing the soluble label needs to remove the lable at last.Protease is commonly used to remove the label,however,the high cost of protease degradation of protein production in enterprises limits its use.Intein is a kind of functional protein which widely distributed in bacteria,fungi and archaea.They can connect two ends of extein together under appropriate conditions.The size of intein is generally between 100 and 600 amino acids,and can be divided into classic intein,micro intein and split intein.With the development of researches on inteins,their applications have become more and more extensive,such as protein semi-synthesis,isotope labeling,cyclic peptide chain and the establishment of new biosensors.In addition,intein can be used together with the purification label in the process of protein purification,and its self-split characteristic can easily remove the label from the purification system,avoiding the effect of the label on the target protein.The application has great value and development prospect.This study combine the intein,soluble labels and purified labels with immunotoxin to realize the soluble expression of immunotoxin in Ecoli,which can simplify the purification procedure of recombinant proteins,avoiding the process of inclusion body denaturation and renaturation,which contains tedious experimental steps and a large number of protein loss.Besides,through the role of tintein,soluble labels and purified labels quickly be removed,which is important for the purification of imminutoxin.MethodsIn this study,the soluble expression of recombinant protein in Ecoli(BL21(DE3),SHuffle T7)cytoplasm was induced by low temperature.Affinity column was used to purify the target protein.The self-split of the intein was achieved by mixture two ends of intein.Ni column was used to remove the impurity of the protein.The protein was highly concentrated by ultrafiltration or Capto L.Protein affinity was detected by FACS or Fortebio.The soluble expression of recombinant protein in Ecoli was explored through three groups of soluble labels,purification labels,and the application of intein and recombinant immunotoxin.At the same time,intein cleavage can easily remove the label from the purification system,which can reduce the experimental cost.In addition,this study also explore the factors affecting the cleavage of intein,which provides references for the development of soluble expression and purification of the immunotoxins.ResultsThe experiments using two kind of inteins include ΔICM and Npu Dna E,two kind of soluble lables include Trx and MBP,two kind of purification labels include His and MBP,two kind of immunotoxins include HA22(sc Fv)-PE38 KDEL and SS1(scds Fv)-PE38 KDEL.They were used to construct recombinant immunotoxin to explore the soluble expression of recombinant protein in Ecoli.In addition,the factors influence the cleavage efficiency of Npu Dna E were investigated.The results showed that different recombinant immunotoxins could achieve soluble expression in Ecoli,and the self-cleavage of intein could remove the soluble label and purification label easily and quickly.The space structure,charge factor,the extein,time,temperature and DTT concentration all had a certain effect on the split efficiency of Npu Dna E.The experimental results provide a new method for the production and purification of recombinant immunotoxin proteins.