Study On The Expression And Purification Technology Of The Candidate Medicinal Recombinant Protein E23sFv-Fdt-tBid Targeting HER2

HER2(Human Epidermal Growth Factor Receptor2) also knowned as ErbB2is frequently overexpressed in gastric cancer, colon cancer, ovarian cancer, lungcancer, breast cancer, prostate cancer, and so on, playing an important role intumorigenesis according to the reports. HER2has become one of the keymolecules in the therapy for HER2positive cancers. HER2-relatedtumorigenesis can be suppressed through blocking the HER2signaling pathwayas well as targeting HER2for antitumor drug delivery.DT (Diphtheria Toxin), a535amino acids exotoxin produced byPseudomonas Aeruginosa, includes fragment A with ribosome-dependentenzyme activity and fragment B composed of receptor-binding domain andtranslocation domain. A translocation peptide kowned as Fdt, The187thto196thamino acids of DT, functions as endosome-cytosol translocation domain with a cleavage site recoganized by furin protease. Consequently, the cleavedC-terminal DT moiety can be relaased to the cytoplasm and exert a biologicaleffect.As a Bcl2family member, Bid is a22kD protein located in the cytoplasm, andcan be cleaved by proteases like caspase8and GrB, and formes a15kDtruncated Bid which can translocate from the cytosol to the mitochondrion. Themitochondrial tBid subsequently causes the release of cytochrome C andactivation of other apoptosis proteins to induce apoptotic cell death.In the previous study, we construct a fusion gene encoding the recombinantprotein of anti-HER2e23scFv, translocation peptide of Fdt and the apoptotictBid. Then the fusion gene was cloned into the prokaryotic expression vectorand expressed from E.coli. We got the highly purified bioactive recombinantprotein e23sFv-Fdt-tBid with His tag after purification and refolding. The invitro study showed that e23sFv-Fdt-tBid could specificially bind to HER2andbe internalized into the cancer cells to induce apoptosis. The invivo studyshowed that the recombinant protein could significantly inhibite tumorigenesisand increase sensitivity of tumors to chemotherapy drugs. The notable tumorinhibition effect of the e23sFv-Fdt-tBid protein indicated that it might be a newcandidate drug for treatment of HER2positive cancers.Nevertheless, the aforementioned studies are limited by a laboratory scalemini-preparation of tumoricidal recombinant protein, precluding the explorationof mature expression and purification technology for a candidate protein drug.In addition, given that the recombinant proteins generated in our previous studyare tagged by His, they didn’t meet the criterion of the clinical drugs generally.Therefore, in this study we attempt to establish the expression and purificationtechnology for the e23sFv-Fdt-tBid recombinant protein in a pharmaceutically applicable tag-free form.We studied the technology of the production and purification of thee23sFv-Fdt-tBid protein expressed by E.coli system. The e23sFv-Fdt-tBid genewas cloned into the pET-22b vector, which was transformed into the E.colistrain BL21(DE3) competent cells. When induced with IPTG, BL21(DE3)cells expressed e23sFv-Fdt-tBid in a low efficiency and generated unexpectedtruncated proteins. After optimization of the codon and the induction conditionsthe production was notably improved but the cells also expressed the protein inthe form of inclusionbodies with truncated proteins. Experiments of the passageof the E.coli showed that it can stably express the recombinant protein. Throughrenaturation of the inclusionbodies using dialysis, dilution and chromatograph,we found it difficult to refold the protein of e23sFv-Fdt-tBid inclusionbodies.We next studied the technology of the expression of the e23sFv-Fdt-tBidprotein by pichia system. The gene of e23sFv-Fdt-tBid was cloned into thevector of PICZα A and then the protein be secreted to the medum by fused withα signal peptide of the pichia cells. The recombinant vector was transformedinto pichia cells by electrotransformation and the positive clones were screenedafter that. SDS-PAGE and Western Blot analysis showed that pichia cells didn’texpress the recombinant protein of e23sFv-Fdt-tBid.We finally studied the expression of the protein of e23sFv-Fdt-tBid in themammalian CHO cells. The e23sFv-Fdt-tBid gene was cloned into the vector ofpcDNA5/FRT and specifically integrated to the genome of CHO cells throughFlp-InTMsystem. After screening with the proper concentration hygromycin Bwe got several positive clones which were confirmed by genome PCR andWestern Blot analysis. The results showed that the recombinant protein wasexpressed correctly with no truncated proteins. In conclusion, we studied the production and purification technology of thee23sFv-Fdt-tBid rencombinant protein and defined the mammalian CHO cells asthe appropriate system for the potein expression. E.cloli strain BL21(DE3) cellsexpressed the protein highly and stably in the form of inclusionbodies difficultto refold. The recombinant protein fused with α signal peptide failed to beexpressed from pichia system according to the SDS-PAGE and Western Blotanalysis. In contrast, the bioactive recombinant protein of e23sFv-Fdt-tBidexpressed correctly from CHO cells and secreted into the medium. Therefore,CHO expression system is a better option for e23sFv-Fdt-tBid though theproduction was low. Taketn together, this study established an appropriateprotein expression system for the e23sFv-Fdt-tBid recombinant protein and thuslaid a foundation for the clinical application of the recombinant protein.