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

The aim of the study is to establish a platform to deliver therapeutic proteins into target cells through a polyarginine-based cell penetrating peptide. To facilitate the expression of therapeutic proteins, a pSUMO (Small Ubiquitin-like Modifier)-R9-EGFP (enhanced green fluorescence protein) prokaryotic expression vector was constructed. After induction, the fusion protein SUMO-R9-EGFP was efficiently expressed. To validate the cell penetrating ability of the fusion protein, HepG2 cells were incubated with the purified R9-EGFP or EGFP protein as control, internalization of the fluorescent proteins was examined by either flow cytometry or confocal microscopy. The result obtained by flow cytometry showed that the R9-EGFP fusion protein could efficiently penetrate into the HepG2 cells in a dose and time-dependent manner. In contrast, the fluorescence was barely detected in the HepG2 cells incubated with EGFP control. The fluorescence intensity of the R9-EGFP treated cells reached plateau phase after 1.5 h. The result obtained by confocal microscopy shows that R9-EGFP efficiently entered into the HepG2 cells and was exclusively located in the cytoplasm, whereas, no fluorescence was detected in the cells incubated with the EGFP control. The heparin inhibition experiment showed that heparin could inhibit penetrating effect of the R9-EGFP protein by about 50%, suggesting that the penetrating ability of the fusion protein is heparin-dependent. In summary, the study has established a platform to deliver therapeutic proteins into target cells through a polyarginine-based penetrating peptide.
Cell-Penetrating Peptides ; biosynthesis ; genetics ; pharmacology ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; biosynthesis ; genetics ; Hep G2 Cells ; Humans ; Peptides ; genetics ; metabolism ; Protein Transport ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology

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

In this study, we expressed and purified supercharged green fluorescent protein (+36GFP) that we used to study its combination with nucleic acid and its cell transduction efficiency as carrier of DNA. We transformed pET+36GFP-HA2 plasmid into Escherichia coli BL21 (DE3), then expressed and purified the target protein. We used the protein to transduce a variety of mammalian cell lines including B16 cells, 293 cells, A549 cells and HepG2 cells at specified protein concentrations. Transduction efficiency of the protein was analyzed by flow cytometry. Under laser scanning confocal microscope, we observed visually transduction efficiency of +36GFP protein (100 nmol/L) to A549 cells. We incubated +36GFP with plasmid DNA and analyzed their binding ability with gel mobility shift assay. Then we transduced cells with the mixture of plasmid DNA/+36GFP protein at various ratio and detected the expression of reporter gene by using laser scanning confocal microscope and flow cytometry. The experimental results demonstrate that +36GFP had high transduction efficiency, and as the concentration increased, the efficiency improved in a dose-dependent manner. Gel mobility shift assay indicates that +36GFP could bind to plasmid DNA, blocking the migration of DNA in the gel in a concentration-dependent manner. After the plasmid wrapped by +36GFP penetrated into cells, the cells could express target protein efficiently, proving that +36GFP had the ability to carry nucleic acids into cells. Sucussful expression and purification of +36GFP protein confirms its high efficiency of cell transduction and its ability as carrier to deliver exogenous nucleic acids into cells.
Cell Line, Tumor ; DNA ; genetics ; metabolism ; Escherichia coli ; genetics ; metabolism ; Green Fluorescent Proteins ; biosynthesis ; genetics ; pharmacology ; Humans ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology ; Transduction, Genetic ; methods ; Transfection

Down syndrome cell adhesion molecule (DSCAM) acts as a netrin-1 receptor and mediates attractive response of axons to netrin-1 in neural development. However, the signaling mechanisms of netrin-DSCAM remain unclear. Here we report that AMP-activated protein kinase (AMPK) interacts with DSCAM through its γ subunit, but does not interact with DCC (deleted in colorectal cancer), another major receptor for netrin-1. Netrin-treatment of cultured cortical neurons leads to increased phosphorylation of AMPK. Both AMPK mutant with dominant-negative effect and AMPK inhibitor can significantly suppress netrin-1 induced neurite outgrowth. Together, these findings demonstrate that AMPK interacts with DSCAM and plays an important role in netrin-1 induced neurite outgrowth. Our study uncovers a previously unknown component, AMPK, in netrin-DSCAM signaling pathway.
AMP-Activated Protein Kinases ; antagonists & inhibitors ; genetics ; metabolism ; Animals ; Cell Adhesion Molecules ; genetics ; metabolism ; Cells, Cultured ; HEK293 Cells ; Humans ; Mice ; Nerve Growth Factors ; pharmacology ; Netrin-1 ; Neurites ; physiology ; Neurons ; cytology ; drug effects ; metabolism ; Phosphorylation ; Protein Binding ; Protein Kinase Inhibitors ; pharmacology ; RNA Interference ; RNA, Small Interfering ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Signal Transduction ; drug effects ; Transfection ; Tumor Suppressor Proteins ; pharmacology

To construct, express and purify Exendin-4 analogue and detect its biological activity in vivo. Insert gene sequence into fusion partner ofpED plasmid which is helped to purification, entitled the new recombinant plasmid 5 Exendin-4 analogue polypeptide gene and fusion partner gene was linked by acid hydrolysisgene, transformed to E. coli BL21 and the fusion protein was induced by lactose. After acid hydrolysis, the Exendin-4 analogue polypeptide separated from fusion chaperon. Anion charge chromatography were used to further purification. 6 to 8 week-old ICR mice were injected (s.c) with Exendin-4 analogue, blood glucose and plasma insulin level was detected in different period after oral glucose tolerance test. The results show that high expression of inclusion body was induced by lactose, which accounted for 40% of germ proteins, the Exendin-4 analogue was obtained with the purity of 91.8% after being purified by anion charge chromatography. Bioactivity assay showed that the level of blood glucose of mouse which treated with exendin-4 analogue was obviously decreased to normal (P < 0.01), and the level of plasma insulin was increased obviously (P < 0.01).
Animals ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Gene Transfer Techniques ; Hypoglycemic Agents ; metabolism ; pharmacology ; Insulin ; blood ; Male ; Mice ; Mice, Inbred ICR ; Peptides ; genetics ; pharmacology ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology ; Venoms ; biosynthesis ; genetics ; pharmacology

OBJECTIVETo clone tenecin gene, an antibacterial peptide gene, from Tenebrio molitor for its prokaryotic expression and explore the molecular mechanism for regulating the expression of antibacterial peptide in Tenebrio molitor larvae.

METHODSThe antibacterial peptide was induced from the larvae of Tenebrio molitor by intraperitoneal injection of Escherichia coli DH-5α (1×10(8)/ml). RT-PCR was performed 72 h after the injection to clone Tenecin gene followed by sequencing and bioinformatic analysis. The recombinant expression vector pET-28a(+)-Tenecin was constructed and transformed into E. coli BL21(DE3) cells and the expression of tenecin protein was observed after IPTG induction.

RESULTSTenecin expression was detected in transformed E.coli using SDS-PAGE after 1 mmol/L IPTG induction. Tenecin gene, which was about 255 bp in length, encoded Tenecin protein with a relative molecular mass of 9 kD. Incubation of E.coli with 80, 60, 40, and 20 µg/ml tenecin for 18 h resulted in a diameter of the inhibition zone of 25.1∓0.03, 20.7∓0.06, 17.2∓0.11 and 9.3∓0.04 mm, respectively.

CONCLUSIONSTenecin protein possesses strong antibacterial activity against E. coli DH-5α, which warrants further study of this protein for its potential as an antibacterial agent in clinical application.

Amino Acid Sequence ; Animals ; Anti-Bacterial Agents ; pharmacology ; Antimicrobial Cationic Peptides ; biosynthesis ; genetics ; Cloning, Molecular ; Escherichia coli ; drug effects ; genetics ; metabolism ; Genetic Vectors ; genetics ; Insect Proteins ; biosynthesis ; genetics ; Larva ; chemistry ; Microbial Sensitivity Tests ; Molecular Sequence Data ; Recombinant Fusion Proteins ; genetics ; isolation & purification ; pharmacology ; Tenebrio ; chemistry

OBJECTIVETo generate a recombinant expression system of repeated serial antibiotic peptide Alloferon-1 DNA segment with trypsin digestion site and to determine its anti-tumor activity in vitro.

METHODSA 14 repeated serial DNA segment of Alloferon-1 with a lysine residual at the C-end that acts as the trypsin digestion site was constructed. pET42a vector and E.coli BL21DE3 were applied to generate the prokaryotic expression system of the repeated serial DNA segment of Alloferon-1. The yield of target recombinant product was measured by SDS-PAGE and Bio-Rad Gel image system. Ni-NTA affinity column, trypsin digestion and Sephadex G-50 column were used to purify 14 rAlloferon-1-K fusion protein and rAlloferon-1-K monomer. By using the co-cultivation of BALB/c mouse splenocyte with K562, KB or SGC tumor cells and CCK-8 detection method, the effects of rAlloferon-1-K, chemosynthetic Alloferon-1 (cAlloferon-1) and Alloferon-1-K (cAlloferon-1-K) on the growth and proliferation of tumor cells were detected.

RESULTSThe prokaryotic expression system E.coli BL21DE3pET42a-14 Alloferon-1-K efficiently expressed 14 rAlloferon-1-K fusion protein under inducement of IPTG,and the yield of fusion protein was approximate 30% of the total bacterial proteins. 0.1≊10 ng/ml rAlloferon-1-K remarkably increased the effect of mouse splenocytes to inhibit the growth and proliferation of K562, KB and SGC cells (P<0.05), and there was no statistically significant difference of the anti-tumor ability of rAlloferon-1-K compared to that of cAlloferon-1 or cAlloferon-1-K (P>0.05).

CONCLUSIONA prokaryotic expression system of repeated serial Alloferon-1 DNA segment has been successfully constructed with high yield of rAlloferon-1-K, which maintains anti-tumor activity in vitro.

Animals ; Antineoplastic Agents ; pharmacology ; Cell Line, Tumor ; Genetic Vectors ; Humans ; Male ; Mice ; Mice, Inbred BALB C ; Peptides ; genetics ; pharmacology ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology

To investigate the effect of hSCGF-alpha on human Umbilical Cord Mesenchymal Stem Cells (hUCMSCs), we obtained hSCGF-alpha using genetic engineering, hSCGF-alpha gene was amplified from hUCMSCs cDNA using two-step PCR and was inserted into pET-28a(+) plasmid vector. Induced by IPTG at 20 degrees Celsius for 24 h, the fusion protein expressed in E. coli BL21 (DE3) was mainly existing in soluble form. The recombinant hSCGF-a was purified using NI-NTA affinity chromatography and the purity was up to 90%. The colony forming test revealed that combined use hSCGF-alpha and rmGM-CSF (recombinant murine GM-colony stimulating factor, rmGM-CSF) had granulocyte/macrophage (GM) promoting effects on murine bone marrow GM progenitor. In addition, the results indicated that hSCGF-alpha and rhGM-CSF had stimulatory effect on hUCMSCs and their synergetic effect was the strongest.
Cell Proliferation ; drug effects ; Cells, Cultured ; Cloning, Molecular ; Drug Synergism ; Escherichia coli ; genetics ; metabolism ; Granulocyte-Macrophage Colony-Stimulating Factor ; pharmacology ; Humans ; Mesenchymal Stromal Cells ; cytology ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology ; Stem Cell Factor ; biosynthesis ; genetics ; Umbilical Cord ; cytology

NB-Cl gene is a potential class IIa bacteriocin gene. To obtain its soluble expression, NB-C1 was fused with the green fluorescent protein (GFP) gene and a recombinant expression vector plVEX 2.4d-GFP-NB-C1 was constructed, which was transformed into Escherichia coli BL21(DE3) pLysS. The expressed fusion protein GFP-NB-CI was purified by Ni-NTA affinity chromatography and the bioactivity was examined using Listeria monocytogenes as the indicator bacteria. The results showed that the expressed fusion protein GFP-NB-C1 was soluble and the final concentration of the purified fusion protein was 36.1 mg/L E. coli culture and had the purity above 95%. The antimicrobial assay of GFP-NB-C1 was analyzed and showed its high activity against Listeria monocytogenes.
Amino Acid Sequence ; Anti-Bacterial Agents ; pharmacology ; Bacteriocins ; biosynthesis ; genetics ; Chromatography, Affinity ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; biosynthesis ; genetics ; Listeria monocytogenes ; drug effects ; Molecular Sequence Data ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology