Bich Hang Do

Soluble expression and partial purification of recombinant human erythropoietin from E. coli

Human erythropoietin (hEpo) is an essential regulator of erythrocyte production that induces the division and differentiation of erythroid progenitor cells in the bone marrow into mature erythrocytes. It is widely used for the treatment of anemia resulting from chronic kidney disease, chemotherapy, and cancer-related therapies. Active hEpo, and hEpo analogs, have been purified primarily from mammalian cells, which has several disadvantages, including low yields and high production costs. Although an Escherichia coli (E. coli) expression system may provide economic production of therapeutic proteins, it has not been used for the production of recombinant hEpo (rhEpo) because it aggregates in inclusion bodies in the E. coli cytoplasm and is not modified post-translationally. We investigated the soluble overexpression of active rhEpo with various protein tags in E. coli, and found out that several tags increased the solubility of rhEpo. Among them maltose binding protein (MBP)-tagged rhEpo was purified using affinity and gel filtration columns. Non-denaturing electrophoresis and MALDI-TOF MS analysis demonstrated that the purified rhEpo had two intra-disulfide bonds identical to those of the native hEpo. An in vitro proliferation assay showed that rhEpo purified from E. coli had similar biological activity as rhEpo derived from CHO cells. Therefore, we report for the first time that active rhEpo was overexpressed as a soluble form in the cytoplasm of E. coli and purified it in simple purification steps. We hope that our results offer opportunities for progress in rhEpo therapeutics.

Crotamine stimulates phagocytic activity by inducing nitric oxide and TNF-α via p38 and NFκ-B signaling in RAW 264.7 macrophages.

Crotamine is a peptide toxin found in the venom of the rattlesnake Crotalus durissus terrificus and has antiproliferative, antimicrobial, and antifungal activities. Herein, we show that crotamine dose-dependently induced macrophage phagocytic and cytostatic activity by the induction of nitric oxide (NO) and tumor necrosis factor-alpha (TNF-α). Moreover, the crotamineinduced expression of iNOS and TNF-α is mediated through the phosphorylation of p38 and the NF-κB signaling cascade in macrophages. Notably, pretreatment with SB203580 (a p38-specific inhibitor) or BAY 11-7082 (an NF-κB inhibitor) inhibited crotamine-induced NO production and macrophage phagocytic and cytotoxic activity. Our results show for the first time that crotamine stimulates macrophage phagocytic and cytostatic activity by induction of NO and TNF-α via the p38 and NF-κB signaling pathways and suggest that crotamine may be a useful therapeutic agent for the treatment of inflammatory disease. [BMB Reports 2016; 49(3): 185-190]

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 β-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.

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.

Soluble expression and purification of bioactive interleukin 33 in E. coli

Interleukin-33 (IL-33) is one of the important alarmins of the immune system and possesses dual functions as an anti- or pro-inflammatory molecule. The production of this cytokine in E. coli is hampered by the insoluble expression in the cytoplasm, resulting in inclusion body formation. In this study, the expression of IL-33 was optimized by fusing the N-terminus of IL-33 with several solubilizing tags that act as chaperones for proper protein folding: maltose binding protein (MBP), b´a´ domain of protein disulfide isomerase (PDIb´a´) and glutathione Stransferase (GST). The expression of the fusion proteins was stimulated by 0.5 mM IPTG at different temperatures, 37, 30, 25, and 18°C. As a result, IL-33 was expressed highly and in soluble form in the cytoplasm of E. coli when fused with MBP or PDIb´a´ tags in the presence of 0.5 mM IPTG at 25 or 30°C. We describe a simple purification procedure of IL-33 from the PDIb´a´-IL-33 construct using immobilized metal affinity chromatography (IMACs) with supplementary of tobacco etch virus (TEV) protease for tag removal. The high bioactivity of purified IL-33 on the proliferation and activation of macrophages was confirmed by MTT and nitrite releasing assays using RAW 264.7 These data show an improved method for producing high grade and yield IL-33.