Antioxidant enzymes fused with cell-penetrating peptides could enter cells and protect cells from irradiation damage. However, the unselective transmembrane ability of cell-penetrating peptide may also bring antioxidant enzymes into tumor cells, thus protecting tumor cells and consequently reducing the efficacy of radiotherapy. There are active matrix metalloproteinase (MMP)-2 or MMP-9 in most tumor cellular microenvironments. Therefore, a fusion protein containing an MMP-2/9 cleavable substrate peptide X, a cell-penetrating peptide R9, a glutathione S-transferase (GST), and a human Cu, Zn superoxide dismutase (SOD1), was designed and named GST-SOD1-X-R9. In the tumor microenvironment, GST-SOD1-X-R9 would lose its cell-penetrating peptide and could not enter tumor cells due to the cleavage of substrate X by active MMP-2/9, thereby achieving selected entering normal cells. The complete nucleotide sequence of SOD1-X-R9 was synthesized and inserted into the prokaryotic expression vector pGEX-4T-1. The pGEX4T-1-SOD1-X-R9 recombinant plasmid was obtained, and soluble expression of the fusion protein was achieved. GST-SOD1-X-R9 was purified by ammonium sulfate precipitation and GST affinity chromatography. The molecular weight of the fusion protein was approximately 47 kDa, consistent with the theoretical value. The SOD and GST activities were 2 954 U/mg and 328 U/mg, respectively. Stability test suggested that almost no change in either SOD activity or GST activity of GST-SOD1-X-R9 was observed under physiological conditions. The fusion protein could be partially digested by collagenase Ⅳ in solution. Subsequently, the effect of MMP-2/9 activity on transmembrane ability of the fusion protein was tested using 2D and 3D cultured HepG2 cells. Little extracellular MMP-2 activity of HepG2 cells was observed under 2D culture condition. While under the 3D culture model, the size and the MMP-2 activity of the HepG2 tumor spheroid increased daily. GST-SOD1-R9 proteins showed the same transmembrane efficiency in 2D cultured HepG2 cells, but the transmembrane efficiency of GST-SOD1-X-R9 in 3D cultured HepG2 spheres was reduced remarkably. This study provided a basis for further investigating the selectively protective effect of GST-SOD1-X-R9 against oxidative damage in normal cells.
Ammonium Sulfate ; Antioxidants ; Cell-Penetrating Peptides/pharmacology* ; Endopeptidases ; Glutathione Transferase/metabolism* ; Humans ; Matrix Metalloproteinase 2/genetics* ; Matrix Metalloproteinase 9/genetics* ; Recombinant Fusion Proteins ; Recombinant Proteins ; Superoxide Dismutase/metabolism* ; Superoxide Dismutase-1

Antimicrobial peptides are the most promising alternatives to antibiotics. However, the strategy of producing antimicrobial peptides by recombinant technology is complicated and expensive, which is not conducive to the large-scale production. Oxysterlin 1 is a novel type of cecropin antimicrobial peptide mainly targeting on Gram-negative bacteria and is of low cytotoxicity. In this study, a simple and cost-effective method was developed to produce Oxysterlin 1 in Escherichia coli. The Oxysterlin 1 gene was cloned into a plasmid containing elastin-like polypeptide (ELP) and protein splicing elements (intein) to construct the recombinant expression plasmid (pET-ELP-I-Oxysterlin 1). The recombinant protein was mainly expressed in soluble form in E. coli, and then the target peptide can be purified with a simple salting out method followed by pH changing. The final yield of Oxysterlin 1 was about 1.2 mg/L, and the subsequent antimicrobial experiment showed the expected antimicrobial activity. This study holds promise for large-scale production of antimicrobial peptides and the in-depth study of its antimicrobial mechanism.
Elastin ; Escherichia coli/genetics* ; Inteins ; Peptides/pharmacology* ; Pore Forming Cytotoxic Proteins ; Recombinant Fusion Proteins/genetics*

OBJECTIVETo observe the effects of recombinant fusion protein interleukin (IL)-18 on the expression of immune-inflammatory factors in the mice infected with Staphylococcus aureus (SA), and to investigate the mechanism of action of IL-18 in defense of SA infection in vivo.

METHODSA total of 40 specific pathogen-free female BLAB/c mice were randomly divided into four groups: control, SA infection, immunized, and intervention. A mouse model of SA infection was established by nasal inoculation with SA liquid. The immunized group and the intervention group were intranasally given IL-18 before SA modeling, and then the SA infection group and the intervention group received the nasal inoculation with SA liquid; the control group was treated with phosphate buffered saline instead. The levels of IL-4, interferon (IFN)-γ, tumor necrosis factor (TNF), granulocyte colony-stimulating factor (G-CSF), IgM in the serum and bronchoalveolar lavage fluid (BALF) of mice were measured by enzyme-linked immunosorbent assay. The expression of macrophage inflammatory protein (MIP)-1α mRNA and MIP-2β mRNA in the lung tissue of mice were determined by real-time fluorescent quantitative PCR.

RESULTSCompared with the control group, the SA infection group and the immunized group had significantly higher levels of IL-4, G-CSF, and IgM in the serum and BALF and expression of MIP-1α mRNA and MIP-2β mRNA in the lung tissue (P<0.05); the SA infection group had a significantly lower level of IFN-γ and a significantly higher level of TNF in the serum and BALF (P<0.05); the immunized group had a significantly higher level of IFN-γ in the serum and BALF (P<0.05). Compared with the SA infection group, the intervention group had significantly higher levels of IL-4, IFN-γ, G-CSF, and IgM in the serum and BALF and expression of MIP-1α mRNA in the lung tissue. In contrast, the intervention group showed a significantly lower level of TNF in the serum and BALF and expression of MIP-2β mRNA in the lung tissue (P<0.05). All the above indicators in the intervention group were significantly higher than those in the control group (P<0.05), except the serum level of IFN-γ.

CONCLUSIONSIn the mice infected with SA, the recombinant fusion protein IL-18 by mucosal immunity can affect inflammatory factors in the serum and BALF and the expression of MIP-1α mRNA and MIP-2β mRNA in the lung tissue to promote the anti-infective immune response and enhance the ability to clear pathogens.

Animals ; Chemokine CCL3 ; analysis ; Female ; Granulocyte Colony-Stimulating Factor ; blood ; Interferon-gamma ; blood ; Interleukin-18 ; therapeutic use ; Interleukin-4 ; blood ; Mice ; Mice, Inbred BALB C ; Recombinant Fusion Proteins ; pharmacology ; therapeutic use ; Staphylococcal Infections ; drug therapy ; immunology

To obtain sufficient purified and active fusion protein-hepatocyte-targeting peptide-human endostatin (HTP-rES), we studied the growth curve and the optimal induction timing of BL21/pET21b-HTP-rES. Different conditions of pH value, induction time, induction concentration and induction temperature were optimized by univariate analysis. After washing, refolding and purifying, the activity of fusion protein was identified by flow cytometry and 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT). Results show that the logarithmic growth phase of BL21/pET21b-HTP-rES was from 1.5 h to 3.5 h, the optimum expression conditions were pH 8.0, 0.06 mmol/L IPTG, at 42 ℃ for 5 h. The purity of inclusion bodies was up to 60% after washing. The purity of target protein was more than 95% after refolding and purification. Our findings provide the foundation for further biological activity and drug development.
Drug Delivery Systems ; Endostatins ; pharmacology ; Escherichia coli ; Hepatocytes ; drug effects ; Humans ; Inclusion Bodies ; Peptides ; pharmacology ; Recombinant Fusion Proteins

Thymopentin (TP5) and bursopentin (BP5) are both immunopotentiators. To explore whether the TP5-BP5 fusion peptide (TBP5) has adjuvant activity or not, we cloned the TBP5 gene and confirmed that the TBP5 gene in a recombinant prokaryotic expression plasmid was successfully expressed in Escherichia coli BL21. TBP5 significantly promoted the proliferation of thymic and splenic lymphocytes of mice. The potential adjuvant activity of the TBP5 was examined in mice by coinjecting TBP5 and H9N2 avian influenza virus (AIV) inactivated vaccine. HI antibody titers, HA antibodies and cytokines levels (IL-4 and IFN-γ) were determined. We found that TBP5 markedly elevated serum HI titers and HA antibody levels, induced the secretion of both IL-4 and IFN-γ cytokines. Furthermore, virus challenge experiments confirmed that TBP5 contributed to inhibition replication of the virus [H9N2 AIV (A/chicken/Jiangsu/NJ07/05)] from mouse lungs. Altogether, these findings suggest that TBP5 may be an effective adjuvant for avian vaccine and that this study provides a reference for further research on new vaccine adjuvants.
Adjuvants, Immunologic ; pharmacology ; Animals ; Antibodies, Viral ; blood ; Cell Proliferation ; drug effects ; Influenza A Virus, H9N2 Subtype ; drug effects ; physiology ; Influenza Vaccines ; immunology ; Interferon-gamma ; immunology ; Interleukin-4 ; immunology ; Lymphocytes ; drug effects ; Mice ; Oligopeptides ; immunology ; Orthomyxoviridae Infections ; drug therapy ; Recombinant Fusion Proteins ; immunology ; Spleen ; cytology ; Thymopentin ; immunology ; Thymus Gland ; cytology ; Vaccines, Inactivated ; immunology ; Virus Replication

To compare the activity of RGD-TRAIL in different expression systems, RGD-TRAIL in both Escherichia coli (E.coli) and Pichia pastoris was constructed and expressed. In vitro activity of RGD-TRAIL from Pichia pastoris expression system was also analyzed. Genetic engineering techniques were used to construct recombinant plasmid pET30-rgd-trail and pHBM-rgd-trail. The recombinant protein RGD-TRAIL was purified with Ni ion affinity chromatography after induction. MTT assay, ELISA, scratch wound healing, transwell migration assay and Hoechst 33342 staining were performed to detect the effects of RGD-TRAIL on proliferation, binding activity, migration and apoptosis. The expression of apoptosis-associated proteins was detected by Western blotting. Recombinant protein RGD-TRAIL was successfully expressed in a form of inclusion body in E.coli, while expressed secretorily in Pichia pastoris. It possessed more potent cytotoxicity than RGD-TRAIL in E.coli by MTT assay. The RGD-TRAIL expressed by Pichia pastoris showed powerful binding affinity with cancer cells expressing α(v), DR4, DR5 and highly potent cytotoxicity through inducing apoptosis of cancer cells. Nuclear fragmentation was examined by Hoechst 33342 staining. Cleaved PARP and caspase-3 were also detected after incubation with RGD-TRAIL. Additionally, RGD-TRAIL inhibited migration significantly in A549 and HT1080 cells. The results demonstrate that Pichia pastoris expression system is more suitable for the recombinant protein RGD-TRAIL. Its binding affinity and antitumor activity might make RGD-TRAIL a promising candidate for cancer therapy.
Antineoplastic Agents ; pharmacology ; Apoptosis ; Blotting, Western ; Cell Line, Tumor ; Chromatography, Affinity ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; Humans ; Oligopeptides ; biosynthesis ; pharmacology ; Pichia ; metabolism ; Plasmids ; Recombinant Fusion Proteins ; biosynthesis ; pharmacology ; TNF-Related Apoptosis-Inducing Ligand ; biosynthesis ; pharmacology

GLP-1 has a variety of anti-diabetic effects. However, native GLP-1 is not suitable for treatment of diabetes due to its short half-life (t½, 2-5 min). Exendin-4 is a polypeptide isolated from lizard saliva, which can bind to GLP-1 receptor, produce physiological effects similar to GLP-1, t½ up to 2.5 h, therefore, we developed a long-lasting GLP-1 receptor agonists and GLP-1-exendin-4 fusion IgG4 Fc [GLP-1-exendin-4/ IgG4(Fc)]. We constructed the eukaryotic expression vector of human GLP-1-exendin-4/IgG4(Fc)-pOptiVEC- TOPO by gene recombination technique and expressed the fusion protein human GLP-1-IgG4 (Fc) in CHO/DG44 cells. The fusion protein stimulated the INS-1 cells secretion of insulin, GLP-1, exendin-4 and fusion protein in CD1 mice pharmacokinetic experiments, as well as GLP-1, exendin-4 and fusion protein did anti-diabetic effect on streptozotocin induced mice. Results demonstrated that the GLP-1-exendin-4/IgG4(Fc) positive CHO/DG44 clones were chosen and the media from these positive clones. Western blotting showed that one protein band was found to match well with the predicted relative molecular mass of human GLP-1-exendin-4/IgG4(Fc). Insulin RIA showed that GLP-1-exendin-4/IgG4(Fc) dose-dependently stimulated insulin secretion from INS-1 cells. Pharmacokinetic studies in CD1 mice showed that with intraperitoneal injection (ip), the fusion protein peaked at 30 min in circulation and maintained a plateau for 200 h. Natural biological half-life of exendin-4 was (1.39 ± 0.28) h, GLP-1 in vivo t½ 4 min, indicating that fusion protein has long-lasting effects on the modulation of glucose homeostasis. GLP-1-exendin-4/IgG4(Fc) was found to be effective in reducing the incidence of diabetes in multiple-low-dose streptozotocin-induced diabetes in mice, longer duration of the biological activity of the fusion protein. The biological activity was significantly higher than that of GLP-1 and exendin-4. GLP-1-exendin-4/IgG4(Fc) has good anti-diabetic activity. It can be used as a long-acting GLP-1 agonists.
Animals ; CHO Cells ; Cricetinae ; Cricetulus ; Diabetes Mellitus, Experimental ; drug therapy ; Glucagon-Like Peptide 1 ; pharmacology ; Glucagon-Like Peptide-1 Receptor ; agonists ; Half-Life ; Humans ; Hypoglycemic Agents ; pharmacology ; Immunoglobulin G ; pharmacology ; Insulin ; secretion ; Mice ; Peptides ; pharmacology ; Recombinant Fusion Proteins ; pharmacology ; Venoms ; pharmacology

Cartilage oligomeric matrix protein-angiopoietin-1 (COMP-Ang1) is an angiogenic factor for vascular angiogenesis. The aim was to investigate the effect of an intracavernosal injection of COMP-Ang1 on cavernosal angiogenesis in a diabetic rat model. Male Otsuka Long-Evans Tokushima Fatty (OLETF) rats made up the experimental group (1 yr old) and Long-Evans Tokushima Otsuka (LETO) rats made up the control group. The experimental group was divided into vehicle only, 10 microg COMP-Ang1, and 20 microg COMP-Ang1. COMP-Ang1 was injected into the corpus cavernosum of the penis. After 4 weeks, the penile tissues of the rats were obtained for immunohistochemistry and Western blot analysis. The immunoreactivity of PECAM-1 and VEGF was increased in the COMP-Ang1 group compared with the vehicle only group. Moreover, the expression of PECAM-1 and VEGF was notably augmented in the 20 microg Comp Ang-1 group. In the immunoblotting study, the expression of PECAM-1 and VEGF protein was significantly less in the OLEFT rats than in the control LETO rats. However, this expression was restored to control level after intracavernosal injection of COMP-Ang1. These results show that an intracavernosal injection of COMP-Ang1 enhances cavernous angiogenesis by structurally reinforcing the cavernosal endothelium.
Angiopoietin-1/genetics/*metabolism ; Animals ; Antigens, CD31/metabolism ; Blood Glucose/analysis ; Blotting, Western ; Body Weight ; Cartilage Oligomeric Matrix Protein/genetics/*metabolism ; Diabetes Mellitus, Experimental/*pathology ; Immunohistochemistry ; Male ; Neovascularization, Physiologic/*drug effects ; Penis/metabolism/pathology ; Rats ; Rats, Long-Evans ; Recombinant Fusion Proteins/biosynthesis/genetics/*pharmacology ; Vascular Endothelial Growth Factor A/metabolism

OBJECTIVETo investigate the effect of Pseudomonas quinolone signal (PQS) on the virulence of Pseudomonas aeruginosa.

METHODSPseudomonas aeruginosa strain PAO1 was treated with PQS alone, PQS plus farnesol, or farnesol alone. The transcriptional levels of the regulator gene ExsA and virulence protein gene ExoS of type III secretion system were examined using quantitative real-time PCR, and spectrophotometry was employed to detect pyocyanin production in the bacteria. The adhesion and invasiveness of the treated PAO1 in cultured alveolar epithelial cells A549 were assessed on plate count agar, and their effects on the survival of a mouse model of peritonitis was compared.

RESULTSThe increase or decrease of PQS did not affect the growth of PAO1. Compared with the untreated bacteria, PQS-treated PAO1 showed obviously increased transcription levels of ExsA and ExoS (P<0.01) and pyocyanin production, which was significantly lowered by farnesol (P<0.01). In A549 cell cultures, farnesol-treated PAO1 exhibited significantly lowered adhesion and invasiveness, while PQS-treated PAO1 caused a significantly decreased survival time of mice with peritonitis (P<0.01). Farnesol treatment did not obviously affected ExsA transcription (P>0.05) but caused a significant reduction in the transcriptional level of Exos (P<0.05) in PAO1. PQS showed no significant effect on the adhesion and invasiveness of PAO1 (P<0.05).

CONCLUSIONPQS can maintain the adhesion and invasiveness of Pseudomonas aeruginosa, and in the hosts of the bacteria, PQS concentration is positively correlated with pyocyanin production and hence negatively with the survival time of the hosts.

ADP Ribose Transferases ; genetics ; metabolism ; Animals ; Bacterial Adhesion ; Bacterial Proteins ; genetics ; metabolism ; Bacterial Toxins ; genetics ; metabolism ; Cell Line ; Humans ; Male ; Mice ; Mice, Inbred BALB C ; Peritonitis ; microbiology ; Pseudomonas aeruginosa ; genetics ; metabolism ; pathogenicity ; Quinolones ; pharmacology ; Recombinant Fusion Proteins ; genetics ; metabolism ; Signal Transduction ; Trans-Activators ; genetics ; metabolism ; Transcription, Genetic ; Virulence

To improve the expression level of tmAMP1m gene from Tenebrio molitor in Escherichia coli, we studied the effects of expression level and activity of the fusion protein HIS-TmAMP1m by conditions, such as culture temperature, inducing time and the final concentration of inductor Isopropyl beta-D-thiogalactopyranoside (IPTG). We analyzed the optimum expression conditions by Tricine-SDS-PAGE electrophoresis, meanwhile, detected its antibacterial activity by using agarose cavity diffusion method. The results suggest that when inducing the recombinant plasmid with a final IPTG concentration of 0.1 mmol/L at 37 degrees C for 4 h, there was the highest expression level of fusion protein HIS-TmAMP1m in Escherichia coli. Under these conditions, the expression of fusion protein accounted for 40% of the total cell lysate with the best antibacterial activity. We purified the fusion protein HIS-TmAMPlm with nickel-nitrilotriacetic acid (Ni-NTA) metal-affinity chromatography matrices. Western blotting analysis indicates that the His monoclonal antibody could be specifically bound to fusion protein HIS-TmAMPlm. After expression by inducing, the fusion protein could inhibit the growth of host cell transformed by pET30a-tmAMP1m. The fusion protein HIS-TmAMP1m had better stability and remained higher antibacterial activities when incubated at 100 degrees C for 10 h, repeated freeze thawing at -20 degrees C, dissolved in strong acid and alkali, or treated by organic solvents and protease. Moreover, the minimum inhibitory concentration results demonstrated that the fusion protein HIS-TmAMP1m has a good antibacterial activity against Staphylococcus aureus, Staphylococcus sp., Corynebacterium glutamicum, Bacillus thuringiensis, Corynebacterium sp. This study laid the foundation to promote the application of insect antimicrobial peptides and further research.
Animals ; Anti-Infective Agents ; pharmacology ; Antimicrobial Cationic Peptides ; biosynthesis ; genetics ; pharmacology ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Insect Proteins ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology ; Tenebrio ; chemistry