Production of recombinant human long-acting IL-18 binding protein: inhibitory effect on ulcerative colitis in mice.

Interleukin-18 binding protein (IL-18BP) is a natural IL-18 inhibitor in vivo, which can effectively neutralize IL-18 and inhibit the inflammatory signaling pathway induced by IL-18, thus playing an anti-inflammatory role. Traditional production methods primarily rely on eukaryotic animal cell expression systems, which often entail complex processes, lower yields, and increase production costs. In this study, we present a novel approach for expressing IL-18BP fusion protein using the Escherichia coli (E. coli) system. The N-terminal segment of IL-18BP was fused with the small ubiquitin-related modifier (SUMO) tag, enabling soluble expression, while the C-terminal segment was fused with the human IgG1 Fc fragment to prolong its in vivo lifespan. Through screening, we obtained a high-expression engineering strain from a single colony and developed optimized protocols for fermentation and purification of the recombinant SUMO-IL-18BP-Fc protein. The SUMO tag was subsequently cleaved using SUMO protease, and the purified recombinant human IL-18BP-Fc (rhIL-18BP-Fc) exhibited a purity exceeding 90% with a yield of 1 g per liter of bacterial solution. The biological activities and underlying mechanisms of rhIL-18BP-Fc were evaluated using cell lines and a mouse model. Our results demonstrated that rhIL-18BP-Fc effectively inhibited IL-18-stimulated IFN-γ production in KG-1a cells in vitro and ameliorated DSS-induced ulcerative colitis in mice. In conclusion, we successfully employed the SUMO fusion system to achieve high-level production, soluble expression, and prolonged activity of rhIL-18BP-Fc in E. coli. These findings lay the groundwork for future large-scale industrial production and pharmaceutical development of rhIL-18BP-Fc protein. KEY POINTS: • Effective expression, fermentation, and purification of bioactive rhIL-18BP-Fc protein in E. coli. • The rhIL-18BP-Fc protein has a great potential for the therapy of ulcerative colitis by inhibiting the expression of inflammatory factors.

Characterization of tangeretin as an activator of nuclear factor erythroid 2-related factor 2/antioxidant response element pathway in HEK293T cells.

Numerous studies have reported that tangeretin is a polymethoxylated flavone with a variety of biological activates, but little research has been done on the antioxidant mechanism of tangeretin. Hence, we investigated the effect of tangeretin on the nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response element (ARE) pathway and its potential molecular mechanisms by in vitro and in silico research. The results of molecular docking suggested that tangeretin bound at the top of the central pore of Kelch-like ECH-associated protein 1 (Keap1) Kelch domain, and the hydrophobic and hydrogen bond interactions contributed to their stable binding. Herein, the regulation of Nrf2-ARE pathway by tangeretin was explored in the human embryonic kidney cell line HEK293T, which is relatively easy to be transfected. Upon binding to tangeretin, Nrf2 translocated to the nucleus of HEK293T cells, which in turn activated the Nrf2-ARE pathway. Luciferase reporter gene analysis showed that tangeretin significantly induced ARE-mediated transcriptional activation. Real-time PCR and Western blot assays showed that tangeretin induced the gene and protein expressions of Nrf2-mediated targets, including heme oxygenase 1 (HO-1), nicotinamide adenine dinucleotide phosphate (NADPH) quinone dehydrogenase 1 (NQO1), and glutamate-cysteine ligase (GCLM). In addition, tangeretin could effectively scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radicals. In summary, tangeretin may be a potential antioxidant via activating the Nrf2-ARE pathway.

Loss of FGFR3 Accelerates Bone Marrow Suppression-Induced Hematopoietic Stem and Progenitor Cell Expansion by Activating FGFR1-ELK1-Cyclin D1 Signaling.

Patients with chemotherapy or radiation therapy often generate anemia and low immunity due to the therapy-induced bone marrow (BM) suppression. To enhance hematopoietic regeneration during the therapy-induced BM suppression urgently need to be solved. Fibroblast growth factors (FGFs) play important regulatory roles in hematopoietic stem and progenitor cell (HSPC) expansion in vitro and in vivo by the FGF receptor (FGFR1-4)-mediated signaling pathway. FGFR3 is an important member of the FGFR family, and its regulatory function in hematopoiesis is largely unknown. Using knockout (KO) mice of FGFR3, we found that loss of FGFR3 does not affect HSPC functions or lineage differentiation during steady-state hematopoiesis, but FGFR3 deletion accelerates HSPC expansion and hematopoiesis recovery via a cell-autonomous manner under 5-fluorouracil-induced BM suppression. Our results showed that FGFR3 inactivation accelerates BM suppression-induced HSPC expansion by upregulating FGFR1 and its downstream transcriptional factor, ELK, which regulates the expression of the cyclin D1 gene at the level of transcription. Further studies confirmed that loss of FGFR3 in hematopoietic cells inhibits in vivo leukemogenesis under BM suppression. Our data found a novel hematopoietic regulatory mechanism by which FGFR3 deletion promotes HSPC expansion under BM suppression and also provided a promising approach to enhance antileukemia and hematopoietic regeneration by inhibiting FGFR3 functions in HSPCs combined with leukemic chemotherapy.

Loss of FGFR3 Delays Acute Myeloid Leukemogenesis by Programming Weakly Pathogenic CD117-Positive Leukemia Stem-Like Cells.

Chemotherapeutic patients with leukemia often relapse and produce drug resistance due to the existence of leukemia stem cells (LSCs). Fibroblast growth factor receptor 3 (FGFR3) signaling mediates the drug resistance of LSCs in chronic myeloid leukemia (CML). However, the function of FGFR3 in acute myeloid leukemia (AML) is less understood. Here, we identified that the loss of FGFR3 reprograms MLL-AF9 (MA)-driven murine AML cells into weakly pathogenic CD117-positive leukemia stem-like cells by activating the FGFR1-ERG signaling pathway. FGFR3 deletion significantly inhibits AML cells engraftment in vivo and extends the survival time of leukemic mice. FGFR3 deletion sharply decreased the expression of chemokines and the prolonged survival time in mice receiving FGFR3-deficient MA cells could be neutralized by overexpression of CCL3. Here we firstly found that FGFR3 had a novel regulatory mechanism for the stemness of LSCs in AML, and provided a promising anti-leukemia approach by interrupting FGFR3.

Construction and characterization of immunoliposomes targeting fibroblast growth factor receptor 3.

Fibroblast growth factor receptor 3 (FGFR3) plays an important regulatory role in tumor cell proliferation and drug resistance. FGFR3 is often constitutively active in many tumors. To deliver drugs into tumor cells by targeting FGFR3 will be a promising and potential strategy for cancer therapy. In this study, a novel fusion protein, ScFv-Cys containing a single chain variable fragment (ScFv) and an additional C-terminal cysteine residue, was generated at a rate of 10 mg/L of bacterial culture and purified at 95% by Ni-NTA chromatography. Subsequently, the recombinant ScFv-Cys was coupled with malPEG2000-DSPE and incorporated into liposomes to generate the immunoliposomes. The results indicated that immunoliposomes can specifically deliver the fluorescent molecules, Dio into bladder cancer cells highly expressing FGFR3. In conclusion, we successfully generated FGFR3-specific immunoliposomes, and proved its targeting effect and delivering ability.

Expression, purification and biological effect of a novel single chain Fv antibody and protamine fusion protein for the targeted delivery of siRNAs to FGFR3 positive cancer cells

Background: Gain-of-function of fibroblast growth factor receptor 3 (FGFR3) is involved in the pathogenesis of many tumors. More and more studies have focused on the potential usage of therapeutic single-chain Fv (ScFv) antibodies against FGFR3. RNA interference (RNAi) has been considered as a promising therapeutic method against cancer. A tool which can deliver small interference RNAs (siRNAs) into FGFR3 positive cancer cells is very promising for anti-tumor therapy. Results: In this study, a novel fusion protein R3P, which consists of FGFR3-ScFv and protamine, was generated in Escherichia coli by inclusion body expression strategy and Ni-NTA chromatography. Its yield reached 10 mg per liter of bacterial culture and its purity was shown to be higher than 95%. 1 µg of R3P could efficiently bind to about 2.5 pmol siRNAs and deliver siRNAs into FGFR3 positive RT112 and K562 cells. Annexin V staining results showed that R3P can deliver the amplified breast cancer 1 (AIB1) siRNAs to induce RT112 cell apoptosis. Conclusion: These results indicated that R3P was a promising carrier tool to deliver siRNAs into FGFR3 positive cancer cells and to exert anti-tumor effect.

Roles of Arf6 in cancer cell invasion, metastasis and proliferation.

ADP-ribosylation factor 6 (Arf6), a member of small GTPases ADP-ribosylation factor (Arf) family, expresses widely in mammalian cells and mainly regulates the functions of membrane traffic and actin remodeling. Current studies indicated that the activation and high expression of Arf6 protein may be significantly correlated with the invasion and metastasis of several tumors, such as breast cancer, pancreatic cancer, lung cancer, etc. Meanwhile, the ability of tumor invasion and metastasis can be suppressed when Arf6 activity is blocked by the inhibitors or small-interfering RNAs of Arf6. To explore the precisely potential mechanisms between Arf6 and the process of tumor invasion, metastasis and proliferation, we concludes the functions and potential signaling pathways of Arf6 in tumor cells and provides an overview about clinical prospects of Arf6 in the screening, diagnosis, treatment and evaluation of prognosis of neoplasms.

5­aza­2'­deoxycytidine promotes migration of acute monocytic leukemia cells via activation of CCL2­CCR2­ERK signaling pathway.

5-aza­2'-deoxycytidine (5-Aza) has been approved for clinical use in the treatment of myelodysplastic syndrome and acute myeloid leukemia (AML). It inhibits cell proliferation and induces cell differentiation by demethylating various genes, including tumor suppressor genes, transcription factors, and genes encoding cell cycle inhibitors. Although it has demonstrated efficacy in the clinic, drug resistance following 5­Aza treatment occurs. Cell migration and invasion following 5­Aza treatment are considered to be the key factors underlying drug resistance; however, there is currently limited information regarding the detailed mechanisms involved. In the present study, the THP­1 monocytic leukemia cell line was employed. The anti­leukemic functions of 5­Aza in THP­1 cells were first investigated. The results demonstrated that 5­Aza induced differentiation and inhibited THP­1 cell growth. Notably, 5­Aza significantly promoted THP­1 cell migration. Using reverse transcription­polymerase chain reaction, Western blot and enzyme­linked immunosorbent assay analyses, 5­Aza treatment was observed to upregulate the expression of chemokine (C­C motif) ligand 2 (CCL2) and C­C chemokine receptor type 2 (CCR2) in THP­1 cells. In addition, the results demonstrated that CCL2 induced extracellular signal­regulated kinase (ERK) phosphorylation by CCR2 in 5­Aza­treated THP­1 cells. Treatment with a CCR2 or ERK inhibitor inhibited the 5­Aza­induced increase in THP­1 cell migration. In conclusion, the results of the present study provide an insight into the molecular mechanism underlying the 5­Aza­induced increase in THP­1 cell migration, as well as a potential strategy to overcome drug resistance in AML therapy.

Raloxifene microsphere-embedded collagen/chitosan/ß-tricalcium phosphate scaffold for effective bone tissue engineering.

Engineering novel scaffolds that can mimic the functional extracellular matrix (ECM) would be a great achievement in bone tissue engineering. This paper reports the fabrication of novel collagen/chitosan/ß-tricalcium phosphate (CCTP) based tissue engineering scaffold. In order to improve the regeneration ability of scaffold, we have embedded raloxifene (RLX)-loaded PLGA microsphere in the CCTP scaffold. The average pore of scaffold was in the range of 150-200µm with ideal mechanical strength and swelling/degradation characteristics. The release rate of RLX from the microsphere (MS) embedded scaffold was gradual and controlled. Also a significantly enhanced cell proliferation was observed in RLX-MS exposed cell group suggesting that microsphere/scaffold could be an ideal biomaterial for bone tissue engineering. Specifically, RLX-MS showed a significantly higher Alizarin red staining indicating the higher mineralization capacity of this group. Furthermore, a high alkaline phosphatase (ALP) activity for RLX-MS exposed group after 15days incubation indicates the bone regeneration capacity of MC3T3-E1 cells. Overall, present study showed that RLX-loaded microsphere embedded scaffold has the promising potential for bone tissue engineering applications.

Expression, Purification and Characterization of Recombinant Canine FGF21 in Escherichia coli.

The canine metabolic diseases, such as obesity and diabetes, have become a worldwide problem. Fibroblast growth factor 21 (FGF21) is a potent regulator which has many biological functions relative to metabolism regulation. It suggests that FGF21 plays important roles in regulating canine metabolic diseases. To acquire the recombinant canine FGF21 (rcFGF21) in Escherichia coli, the recombinant bacteria were induced by 0.5 mM IPTG for 16 hours at 16 °C, and the rcFGF21 protein was purified by Ni-NTA. 8 mg rcFGF21 was acquired from one liter bacteria. The rcFGF21 protein has specific immunoblot reactivity against anti-FGF21 and anti-His antibody. The in vivo experimental result showed that rcFGF21 can significantly reduce plasma glucose of STZ-induced diabetic mice.

A novel uPAg-KPI fusion protein inhibits the growth and invasion of human ovarian cancer cells in vitro.

Urokinase-type plasminogen activator (uPA) acts by breaking down the basement membrane and is involved in cell proliferation, migration and invasion. These actions are mediated by binding to the uPA receptor (uPAR) via its growth factor domain (GFD). The present study evaluated the effects of uPAg-KPI, a fusion protein of uPA-GFD and a kunitz protease inhibitor (KPI) domain that is present in the amyloid ß-protein precursor. Using SKOV-3 cells, an ovarian cancer cell line, we examined cell viability, migration, invasion and also protein expression. Furthermore, we examined wound healing, and migration and invasion using a Transwell assay. Our data showed that uPAg-KPI treatment reduced the viability of ovarian cancer SKOV-3 cells in both a concentration and time-dependent manner by arresting tumor cells at G1/G0 phase of the cell cycle. The IC50 of uPAg-KPI was 0.5 µg/µl after 48 h treatment. At this concentration, uPAg-KPI also inhibited tumor cell colony formation, wound closure, as well as cell migration and invasion capacity. At the protein level, western blot analysis demonstrated that uPAg-KPI exerted no significant effect on the expression of total extracellular signal-regulated kinase (ERK)1/ERK2 and AKT, whereas it suppressed levels of phosphorylated ERK1/ERK2 and AKT. Thus, we suggest that this novel uPAg-KPI fusion protein reduced cell viability, colony formation, wound healing and the invasive ability of human ovarian cancer SKOV-3 cells in vitro by regulating ERK and AKT signaling. Further studies using other cell lines will confirm these findings.

ALDH2 gene polymorphism in different types of cancers and its clinical significance.

Aldehyde dehydrogenase 2 (ALDH2), an important mitochondrial enzyme governing ethanol metabolism, displays polymorphism in human. Recent evidence suggested that genetic polymorphism in ALDH2 gene may be significantly correlated with the susceptibility to cancer, such as colorectal cancer, esophageal cancer, and liver cancer. To investigate the correlation between ALDH2 mutant gene and the risk of a certain cancer, many studies have been done by testing the ALDH2 genotype in patients with cancers. Here, we summarized 84 ALDH2 gene single nucleotide polymorphism (SNP) sites in human cancer, which focus primarily on the rs671 SNP site. As a novel biological marker, ALDH2 displays a very attractive prospect in the screening, diagnosis and evaluation of the prognosis of many diseases. Moreover, much attention has been attracted to the studies of the biological functions and potential value of ALDH2 in the human cancer treatment. This review will provide an overview about the clinical prospects of ALDH2 based on the available information.

Expression and purification of soluble single-chain Fv against human fibroblast growth factor receptor 3 fused with Sumo tag in Escherichia coli

Background Overexpression or mutated activation of Fibroblast growth factor receptor 3 (FGFR3) is involved in the pathogenesis of many tumors. More and more studies focus on the potential usage of therapeutic antibodies against FGFR3. Results In this study, a novel single-chain Fv (ScFv) against FGFR3 was prepared and characterized. To achieve the soluble expression, ScFv was fused with Sumo (Small ubiquitin-related modifier) by polymerase chain reaction (PCR), and cloned into pET-20b. The recombinant bacteria were induced by 0.5 mM Isopropyl-ß-d-thiogalactopyranoside (IPTG) for 16 h at 20°C, and the supernatant liquid of Sumo-ScFv was harvested and purified by Ni-NTA chromatography. After being cleaved by the Sumo protease, the recombinant ScFv was released from the fusion protein, and further purified by Ni-NTA chromatography. The purity of ScFv was shown to be higher than 95% and their yield reached 4 mg per liter of bacterial culture. In vitro data showed that ScFv can significantly attenuate FGF9-induced phosphorylation of FGFR3. Conclusion We provide a novel method to produce soluble expression and bioactive functions of ScFv in Escherichia coli.

CD123 and its potential clinical application in leukemias.

The α chain of interleukin 3 receptor (IL-3Rα or CD123), together with the common ß (ßc) subunit, forms a high-affinity IL-3R with biological function. In recent years, emerging research has found that CD123 is highly expressed on the surface of various cells (e.g., leukemia stem cells, LSCs), and it is associated with the initiation and development of many diseases, such as acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). As a novel biological marker, it has an attractive prospect in the diagnosis, targeted therapy, and evaluation of prognosis of many diseases. For these reasons, much attention has been attracted to the studies of the biological functions and potential value in the clinical application of CD123. In this review, the clinical prospects of CD123 will be discussed on the basis of information available.

Co-inhibition of NF-κB and JNK is synergistic in TNF-expressing human AML.

Leukemic stem cells (LSCs) isolated from acute myeloid leukemia (AML) patients are more sensitive to nuclear factor κB (NF-κB) inhibition-induced cell death when compared with hematopoietic stem and progenitor cells (HSPCs) in in vitro culture. However, inadequate anti-leukemic activity of NF-κB inhibition in vivo suggests the presence of additional survival/proliferative signals that can compensate for NF-κB inhibition. AML subtypes M3, M4, and M5 cells produce endogenous tumor necrosis factor α (TNF). Although stimulating HSPC with TNF promotes necroptosis and apoptosis, similar treatment with AML cells (leukemic cells, LCs) results in an increase in survival and proliferation. We determined that TNF stimulation drives the JNK-AP1 pathway in a manner parallel to NF-κB, leading to the up-regulation of anti-apoptotic genes in LC. We found that we can significantly sensitize LC to NF-κB inhibitor treatment by blocking the TNF-JNK-AP1 signaling pathway. Our data suggest that co-inhibition of both TNF-JNK-AP1 and NF-κB signals may provide a more comprehensive treatment paradigm for AML patients with TNF-expressing LC.

High-level expression, purification, and characterization of bifunctional ScFv-9R fusion protein.

Fibroblast growth factor receptor 3 (FGFR3) is a noted proto-oncogene involved in the pathogenesis of many tumors, so more and more studies focus on the potential use of receptor kinase inhibitor and therapeutic antibodies against FGFR3. In this study, we designed a novel fusion protein containing the single-chain Fv (ScFv) against FGFR3 and 9-arginine, denoted as ScFv-9R. To achieve the high-level production and soluble expression, ScFv and ScFv-9R were fused with small ubiquitin-related modifier (Sumo) by polymerase chain reaction and expressed in Escherichia coli BL21 (DE3). The recombinant bacteria was induced by 0.5 mM isopropyl-ß-D-thiogalactopyranoside for 20 h at 20 °C; supernatants of Sumo-ScFv was harvested and purified by DEAE Sepharose FF and Ni-NTA orderly, and supernatants of Sumo-ScFv-9R was harvested and purified by Ni-NTA. After cleaved by the Sumo protease, the recombinant ScFv or ScFv-9R was released from the fusion protein, respectively. The purity of ScFv or ScFV-9R was shown to be higher than 90 %, and their yield reached 3-5 mg per liter of bacterial culture. In vitro data showed that ScFV-9R can attenuate the phosphorylation of FGFR3 and ERK in the absence or presence of FGF9. Gel retardation assay showed that 1 µg of ScFv-9R could efficiently bind to about 4 pmol siRNA. Fluorescent microscope analysis showed that ScFv-9R can efficiently bind and deliver siRNA into RT112 cells. In conclusion, we use Sumo fusion system to acquire high-level production, soluble expression, and bifunctional activity of ScFv-9R in E. coli. Our results also revealed that ScFv-9R, as a novel carrier, may have potential applications in antitumor studies and pharmaceutical development.

TNF-α/Fas-RIP-1-induced cell death signaling separates murine hematopoietic stem cells/progenitors into 2 distinct populations.

We studied the effects of TNF-α and Fas-induced death signaling in hematopoietic stem and progenitor cells (HSPCs) by examining their contributions to the development of bone marrow failure syndromes in Tak1-knockout mice (Tak1(-/-)). We found that complete inactivation of TNF-α signaling by deleting both of its receptors, 1 and 2 (Tnfr1(-/-)r2(-/-)), can prevent the death of 30% to 40% of Tak1(-/-) HSPCs and partially repress the bone marrow failure phenotype of Tak1(-/-) mice. Fas deletion can prevent the death of 5% to 10% of Tak1(-/-) HSPCs but fails to further improve the survival of Tak1(-/-)Tnfr1(-/-)r2(-/-) HSPCs, suggesting that Fas might induce death within a subset of TNF-α-sensitive HSPCs. This TNF-α/Fas-induced cell death is a type of receptor-interacting protein-1 (RIP-1)-dependent programmed necrosis called necroptosis, which can be prevented by necrostatin-1, a specific RIP-1 inhibitor. In addition, we found that the remaining Tak1(-/-) HSPCs died of apoptosis mediated by the caspase-8-dependent extrinsic apoptotic pathway. This apoptosis can be converted into necroptosis by the inhibition of caspase-8 and prevented by inhibiting both caspase-8 and RIP-1 activities. We concluded that HSPCs are heterogeneous populations in response to death signaling stimulation. Tak1 mediates a critical survival signal, which protects against both TNF-α/Fas-RIP-1-dependent necroptosis and TNF-α/Fas-independent apoptosis in HSPCs.

Transforming growth factor-beta-activated kinase 1 regulates natural killer cell-mediated cytotoxicity and cytokine production.

Carma1, a caspase recruitment domain-containing membrane-associated guanylate kinase, initiates a unique signaling cascade via Bcl10 and Malt1 in NK cells. Carma1 deficiency results in reduced phosphorylation of JNK1/2 and activation of NF-κB that lead to impaired NK cell-mediated cytotoxicity and cytokine production. However, the precise identities of the downstream signaling molecules that link Carma1 to these effector functions were not defined. Here we show that transforming growth factor-ß (TGF-ß)-activated kinase 1 (TAK1) is abundantly present in NK cells, and activation via NKG2D results in its phosphorylation. Lack of Carma1 considerably reduced TAK1 phosphorylation, demonstrating the dependence of TAK1 on Carma1 in NKG2D-mediated NK cell activations. Pharmacological inhibitor to TAK1 significantly reduced NK-mediated cytotoxicity and its potential to generate IFN-γ, GM-CSF, MIP-1α, MIP-1ß, and RANTES. Conditional in vivo knockdown of TAK1 in NK cells from Mx1Cre(+)TAK1(fx/fx) mice resulted in impaired NKG2D-mediated cytotoxicity and cytokine/chemokine production. Inhibition or conditional knockdown of TAK1 severely impaired the NKG2D-mediated phosphorylation of ERK1/2 and JNK1/2 and activation of NF-κB and AP1. Our results show that TAK1 links Carma1 to NK cell-mediated effector functions.

The effect of transforming growth factor-beta1 on nasopharyngeal carcinoma cells: insensitive to cell growth but functional to TGF-beta/Smad pathway.

OBJECTIVES: This study explored the response of nasopharyngeal carcinoma cells to TGF-beta1-induced growth suppression and investigated the roles of the TGF-beta/Smad signaling pathway in nasopharyngeal carcinoma cells. METHODS: The cells of nasopharyngeal carcinoma cell line CNE2 were treated with TGF-beta1. The growth responses of CNE2 cells were analyzed by MTT assay. The mRNA expression and protein subcellular localization of the TGF-beta/Smad signaling components in the CNE2 were determined by real time RT-PCR and immunocytochemical analysis. RESULTS: We found that the growth of CNE2 cells was not suppressed by TGF-beta1. The signaling proteins TbetaRII, Smad 7 were expressed normally, while Smad2, Smad3, and Smad4 increased significantly at the mRNA level. TGF-beta type II receptor and Smad7 had no change compared to the normal nasopharyngeal epithelial cells. In addition, Smad2 was phosphorylated to pSmad2, and the activated pSmad2 translocated into the nucleus from the cytoplasm, while the inhibitory Smad-Smad7 translocated from the nucleus to the cytoplasm after TGF-beta1 stimulation. CONCLUSION: The results suggested that CNE2 cells are not sensitive to growth suppression by TGF-beta1, but the TGF-beta/Smad signaling transduction is functional. Further work is needed to address a more detailed spectrum of the TGF-beta/Smad signaling pathway in CNE2 cells.

High-level expression and purification of soluble recombinant FGF21 protein by SUMO fusion in Escherichia coli.

BACKGROUND: Fibroblast growth factor 21 (FGF21) is a promising drug candidate to combat metabolic diseases. However, high-level expression and purification of recombinant FGF21 (rFGF21) in Escherichia coli (E. coli) is difficult because rFGF21 forms inclusion bodies in the bacteria making it difficult to purify and obtain high concentrations of bioactive rFGF21. To overcome this problem, we fused the FGF21 with SUMO (Small ubiquitin-related modifier) by polymerase chain reaction (PCR), and expressed the fused gene in E. coli BL21(DE3). RESULTS: By inducing with IPTG, SUMO-FGF21 was expressed at a high level. Its concentration reached 30% of total protein, and exceeded 95% of all soluble proteins. The fused protein was purified by DEAE sepharose FF and Ni-NTA affinity chromatography. Once cleaved by the SUMO protease, the purity of rFGF21 by high performance liquid chromatography (HPLC) was shown to be higher than 96% with low endotoxin level (<1.0 EU/ml). The results of in vivo animal experiments showed that rFGF21 produced by using this method, could decrease the concentration of plasma glucose in diabetic rats by streptozotocin (STZ) injection. CONCLUSIONS: This study demonstrated that SUMO, when fused with FGF21, was able to promote its soluble expression of the latter in E. coli, making it more convenient to purify rFGF21 than previously. This may be a better method to produce rFGF21 for pharmaceutical research and development.