Design,Synthesis And Activity Of Anti-tumor Bioactive Chimeric Proteins

Over the past decades,the increasing resistance of cancer cells to chemotherapeutic agents,coupled with harmful side effects,has increased the urgency of developing new anti-cancer drugs.Compared with conventional radiotherapy,peptide-based targeted tumor therapy has the advantages of high specificity and low side effects.To improve the problem of poor stability of anticancer lytic peptides while enhancing their tumor targeting,we chose chimeric peptide design to achieve this purpose.In this study,we used the cyclotide MCoTI-I as a carrier for the chimeric peptide.We expressed the natural trypsin inhibitor cyclotides MCoTI-I in Escherichia coli(E.coli)using intein-mediated protein trans-splicing,which was evaluated and shown to have trypsin inhibitory activity,high thermal and serum stability,low cytotoxicity,and cell permeability,and could be used as an excellent peptide drug carrier framework.Then,using MCoTI-I as a framework,we designed the anti-cancer chimeric cyclotide MCoGPx1-B2 by grafting the anticancer active peptide GPx1-B2 into loop 6 of MCoTI-I.The structural sequence of the anti-cancer chimeric cyclotide MCoGPx1-B2 was designed and expressed to improve the stability and cell membrane penetration of antitumor polypeptides.The evaluation showed that the anti-cancer chimeric cyclotide MCoGPx1-B2 still possessed trypsin inhibitory activity and thermal stability.In the second part of the study,we designed the anti-tumor bioactive chimeric protein anti-EGFR husc Fv-ZXR2 by using the humanized single-chain antibody husc Fv of cetuximab as the targeting vector and fusing it with the anticancer lytic peptides ZXR-2,and obtained high purity complexed proteins by purifying and complexing the inclusion bodies.The results of cytotoxicity,serum stability,live-dead cell staining and fluorescent dye labeling experiments showed that the designed chimeric protein anti-EGFR husc Fv-ZXR2 not only retained the target recognition function of anti-EGFR husc Fv for EGFR,but also retained the property of peptide ZXR-2to kill tumor cells by cleaving the cell membrane of tumor cells,with The dual anti-tumor effect can overcome the possible drug resistance in clinical practice.This sc Fv-ACLP fusion protein has good stability and powerful anticancer activity,and also has a biphasic effect on cancer therapy,because ZXR-2represents a rapid action on cancer cells,while anti-EGFR husc Fv can have a long-term inhibition of cancer cell proliferation.In summary,two peptide drug frameworks were used as carriers to construct chimeric anti-cancer bioactive proteins,and biological expression methods were used to produce chimeric anti-cancer bioactive proteins with tumor targeting and good stability,providing a new idea to improve the stability and targeting function of anti-cancer peptides.