Crotamine-based recombinant immunotoxin targeting HER2 for enhanced cancer cell specificity and cytotoxicity.

Crotamine, one of the major toxins present in the venom of the South American rattlesnake Crotalus durissus terrificus, exhibits potent cytotoxic properties and has been suggested for cancer therapy applications. However, its selectivity for cancer cells needs to be improved. This study designed and produced a novel recombinant immunotoxin, HER2(scFv)-CRT, composed of crotamine and single-chain Fv (scFv) derived from trastuzumab targeting human epidermal growth factor receptor 2 (HER2). The recombinant immunotoxin was expressed in Escherichia coli and purified using various chromatographic techniques. The cytotoxicity of HER2(scFv)-CRT was assessed in three breast cancer cell lines, demonstrating enhanced specificity and toxicity in HER2-expressing cells. These findings suggest that the crotamine-based recombinant immunotoxin has the potential to expand the repertoire of recombinant immunotoxin applications in cancer therapy.

Prokaryotic soluble overexpression and purification of oncostatin M using a fusion approach and genetically engineered E. coli strains.

Human Oncostatin M (OSM), initially discovered as a tumour inhibitory factor secreted from U-937 cells, is a gp130 (IL-6/LIF) cytokine family member that exhibits pleiotropic effects in inflammation, haematopoiesis, skeletal tissue alteration, liver regeneration, cardiovascular and metabolic diseases. Cytoplasmic expression of OSM in Escherichia coli results in inclusion bodies, and complex solubilisation, refolding and purification is required to prepare bioactive protein. Herein, eight N-terminal fusion variants of OSM with hexahistidine (His6) tag and seven solubility-enhancing tags, including thioredoxin (Trx), small ubiquitin-related modifier (Sumo), glutathione S-transferase (GST), maltose-binding protein (MBP), N-utilisation substance protein A (Nusa), human protein disulphide isomerase (PDI) and the b'a' domain of PDI (PDIb'a'), were tested for soluble OSM expression in E. coli. The His6-OSM plasmid was also introduced into genetically engineered Origami 2 and SHuffle strains to test expression of the protein. At 18 °C, MBP-tagged OSM was highly expressed and solubility was dramatically enhanced. In addition, His6-OSM was more highly expressed and soluble in Origami 2 and SHuffle strains than in BL21(DE3). MBP-OSM and His6-OSM were purified more than 95% with yields of 11.02 mg and 3.27 mg from a 500 mL culture. Protein identity was confirmed by mass spectroscopy, and bioactivity was demonstrated by in vitro inhibition of Th17 cell differentiation.

Prokaryotic soluble expression and purification of bioactive human fibroblast growth factor 21 using maltose-binding protein.

Human fibroblast growth factor 21 (hFGF21) has been characterized as an important regulator of glucose and lipid metabolism homeostasis. Here, to produce hFGF21 efficiently in Escherichia coli, the expression and solubility of hFGF21 were tested and optimised by fusing the protein with one of eight tags: hexahistidine (His6), thioredoxin (Trx), small ubiquitin-related modifier (Sumo), glutathione S-transferase (GST), maltose-binding protein (MBP), N-utilisation substance protein A (NusA), human protein disulphide isomerase (PDI), and the b'a' domain of PDI (PDIb'a'). Each tag increased solubility of the protein when the expression temperature was 18°C. Unlike many other tags that were tested, MBP significantly enhanced the solubility of the protein also in the culture condition at 37°C. Thus, the MBP-hFGF21 construct was further pursued for optimisation of affinity chromatography purification. After tag removal, 8.1 mg of pure hFGF21 was obtained as a final product from 500 mL of starting culture. The protein was then characterised by mass spectroscopy and an in vitro functional assay using NIH-3T3 cells transfected with a ß-klotho reporter gene. These characteristics are similar to those of commercial hFGF21. Thus, the MBP tag is useful for efficient prokaryotic production and purification of bioactive hFGF21.

Soluble Prokaryotic Expression and Purification of Bioactive Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is considered as an antitumor agent owing to its ability to induce apoptosis of cancer cells without imparting toxicity toward most normal cells. TRAIL is produced in poor yield because of its insoluble expression in the cytoplasm of E. coli. In this study, we achieved soluble expression of TRAIL by fusing maltose-binding protein (MBP), b'a' domain of protein disulfide isomerase (PDIb'a'), or protein disulfide isomerase at the N-terminus of TRAIL. The TRAIL was purified using subsequent immobilized metal affinity chromatography and amylose-binding chromatography, with the tag removal using tobacco etch virus protease. Approximately 4.5 mg of pure TRAIL was produced from 125 ml flask culture with a purification yield of 71.6%. The endotoxin level of the final product was 0.4 EU/µg, as measured by the Limulus amebocyte lysate endotoxin assay. The purified TRAIL was validated and shown to cause apoptosis of HeLa cells with an EC50 and Hill coefficient of 0.6 ± 0.03 nM and 2.41 ± 0.15, respectively. The high level of apoptosis in HeLa cells following administration of purified TRAIL indicates the significance and novelty of this method for producing high-grade and high-yield TRAIL.

Granulocyte colony-stimulating factor (GCSF) fused with Fc Domain produced from E. coli is less effective than Polyethylene Glycol-conjugated GCSF.

Human granulocyte colony-stimulating factor (GCSF) is a well-known cytokine for neutropenia treatment. However, daily injections are required due to the short circulating half-life of the protein. To overcome this bottleneck, we fused GCSF with the Fc domain of IgG1 at the C terminus (GCSF-Fc) and with the maltose binding protein (MBP) tag at the N-terminus and expressed it as a soluble protein in the cytoplasm of E. coli. We also conjugated PEG aldehyde to GCSF to make PEG-GCSF. The bioactivities of GCSF-Fc and PEG-GCSF were similar to native GCSF using the mouse M-NFS-60 myelogenous leukemia cell line. The EC50 dose-response curves for GCSF, GCSF-Fc and PEG-GCSF were 37 ± 12 pM, 75 ± 13.5 pM and 46 ± 5.5 pM, respectively. When the proteins were injected into neutropenic rats, the group injected with PEG-GCSF showed the highest and fastest recovery of neutrophils, followed by GCSF-Fc and GCSF. ELISA assay revealed the PEG-GCSF had the longest plasma circulation (>72 h), followed by GCSF-Fc (>48 h) and GCSF (~24 h), which is consistent with the in vivo activities of the proteins. In summary, the GCSF-Fc purified from E. coli was not as efficient as PEG-GCSF in treating neutropenic rats.