Soluble Cytoplasmic Expression and Purification of Immunotoxin HER2(scFv)-PE24B as a Maltose Binding Protein Fusion.
Sangsu ParkMinh Quan NguyenHuynh Kim Khanh TaMinh Tan NguyenGunsup LeeChong Jai KimYeon-Jin JangHan ChoePublished in: International journal of molecular sciences (2021)
Human epidermal growth factor receptor 2 (HER-2) is overexpressed in many malignant tumors. The anti-HER2 antibody trastuzumab has been approved for treating HER2-positive early and metastatic breast cancers. Pseudomonas exotoxin A (PE), a bacterial toxin of Pseudomonas aeruginosa, consists of an A-domain with enzymatic activity and a B-domain with cell binding activity. Recombinant immunotoxins comprising the HER2(scFv) single-chain Fv from trastuzumab and the PE24B catalytic fragment of PE display promising cytotoxic effects, but immunotoxins are typically insoluble when expressed in the cytoplasm of Escherichia coli, and thus they require solubilization and refolding. Herein, a recombinant immunotoxin gene was fused with maltose binding protein (MBP) and overexpressed in a soluble form in E. coli. Removal of the MBP yielded stable HER2(scFv)-PE24B at 91% purity; 0.25 mg of pure HER2(scFv)-PE24B was obtained from a 500 mL flask culture. Purified HER2(scFv)-PE24B was tested against four breast cancer cell lines differing in their surface HER2 level. The immunotoxin showed stronger cytotoxicity than HER2(scFv) or PE24B alone. The IC50 values for HER2(scFv)-PE24B were 28.1 ± 2.5 pM (n = 9) and 19 ± 1.4 pM (n = 9) for high HER2-positive cell lines SKBR3 and BT-474, respectively, but its cytotoxicity was lower against MDA-MB-231 and MCF7. Thus, fusion with MBP can facilitate the soluble expression and purification of scFv immunotoxins.
Keyphrases
- binding protein
- epidermal growth factor receptor
- escherichia coli
- pseudomonas aeruginosa
- squamous cell carcinoma
- air pollution
- particulate matter
- endothelial cells
- advanced non small cell lung cancer
- small cell lung cancer
- stem cells
- cystic fibrosis
- breast cancer cells
- nitric oxide
- cell proliferation
- cell therapy
- single cell
- risk assessment
- mesenchymal stem cells
- genome wide
- drug resistant
- young adults
- metastatic breast cancer
- water soluble
- staphylococcus aureus
- acinetobacter baumannii
- bone marrow
- cell death
- copy number
- plant growth