PURPOSE: SNF2L belongs to Imitation Switch family and plays an essential role in neural tissues and gonads. In our previous studies, we have demonstrated that the basal transcription of human SNF2L gene is regulated by two cis-elements, cAMP response element (CRE)- and Sp1-binding sites. Recent studies suggested that cyclic adenosine monophosphate (cAMP) stimulation significantly up-regulated SNF2L expression in ovarian granulose cells. These data suggested that protein kinase-mediated signal pathways might also regulate SNF2L expression in neural cells. We therefore investigated the effects of agents that activate protein kinases A on SNF2L gene expression in neural cells. MATERIALS AND METHODS: To increase intracellular cAMP levels, all neural cells were treated with forskolin and dbcAMP, two cAMP response activators. We exmined the effects of cAMP on the promoter activity of human SNF2L gene by luciferase reporter gene assays, and further examined the effects of cAMP on endogenous SNF2L mRNA levels by qPCR. RESULTS: Transient expression of a luciferase fusion gene under the control of the SNF2L promoter was significantly increased by treatment of rat primary neurons with forskolin or dbcAMP, but not PC12, C6 and SH-SY5Y cells. Consistently, treatment with forskolin or dbcAMP could enhance endogenous SNF2L mRNA levels also only in rat primary neurons. CONCLUSION: These results suggest that the CRE consensus sequence in the SNF2L proximal promoter most likely confers constitutive activation and regulation by cAMP in neural cells.
Animals ; Bucladesine/pharmacology ; Cell Line ; Colforsin/pharmacology ; Cyclic AMP/*metabolism ; DNA-Binding Proteins/chemistry/*genetics/metabolism ; *Gene Expression Regulation ; Humans ; Luciferases/analysis ; Neurons/*metabolism ; PC12 Cells ; Promoter Regions, Genetic ; RNA, Messenger/metabolism ; Rats ; Rats, Wistar ; Recombinant Fusion Proteins/analysis ; *Response Elements ; Transcription Factors/chemistry/*genetics/metabolism

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

Follicle-stimulating hormone (FSH) is a glycoprotein which regulates the development, growth, pubertal maturation and reproductive processes of the body. Exogenous FSH has been used to promote ovarian follicular growth and maturation in female and spermatogenesis in male. The relative short elimination half life and rapid metabolic clearance of current versions of FSH require a daily or twice-daily scheduled subcutaneous injection to maintain stable FSH level being not below the threshold during ovarian stimulation. The development of recombinant long-acting FSH with enhanced biological activities may be helpful for less injection therefore to improve patient compliance, while reducing patient stress and error rates. A number of technological strategies have been explored to develop recombinant longer-acting FSH. For examples, attachment of the C-terminal peptide (CTP) of the human chorionic gonadotropin beta subunit or a sequence containing potential glycosylation sites to either subunit of FSH, creation of a single chain containing the alpha and beta subunits of FSH combined with CTP or N-linked glycosylation signal sequence as a linker, or fusion of the Fc domain of IgGi to FSH. Based on the modifiable molecular structure and pharmacokinetic and pharmacodynamic properties of recombinant FSH, it is hopeful that more FSH drugs with prolonged half-life and increased bioactivity will be developed to meet the modern clinical demands.
Animals ; Follicle Stimulating Hormone, Human ; chemistry ; genetics ; metabolism ; pharmacology ; Glycosylation ; Half-Life ; Humans ; Immunoglobulin Fc Fragments ; chemistry ; metabolism ; Ovulation Induction ; methods ; Receptors, FSH ; chemistry ; metabolism ; Recombinant Fusion Proteins ; chemistry ; genetics ; metabolism ; pharmacology ; Reproduction ; drug effects

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

With the genomic DNA of strain EJS-3 as the template, we amplified the gene of fibrinolytic enzyme from Paenibacillus polymyxa (PPFE-I) by PCR. We purified the PCR product and ligated it into pMD19-T. After DNA sequencing, we cloned the PPFE-I gene into expression vector pET-DsbA and transformed it into Escherichia coli BL21(DE3). Upon induction of IPTG, we found that the activity of recombinant fibrinolytic enzyme fused with DsbA expressed in Escherichia coli was 228 IU/mL. SDS-PAGE analysis showed that the recombinant enzyme was soluble and accounted for about 18.4% of total cell protein. Western blotting demonstrated that the recombinant protein was DsbA-PPFE-I. We purified the recombinant enzyme by Ni affinity chromatography, thrombin digestion and sephadex G-100 gel-filtration, and identified the molecular weight of purified product to be 66.3 kDa with MALDI-TOF mass spectrometry. The purified enzyme exhibited distinct fibrinolytic activity on fibrin plate.
Antifibrinolytic Agents ; pharmacology ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Fibrinolytic Agents ; metabolism ; Genetic Vectors ; genetics ; Paenibacillus ; chemistry ; enzymology ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology

Using directed mutagenesis and phage display on a soluble fragment of the human immunoglobulin super-family receptor ILT2 (synonyms: LIR1, MIR7, CD85j), we have selected a range of mutants with binding affinities enhanced by up to 168,000-fold towards the conserved region of major histocompatibility complex (MHC) class I molecules. Produced in a dimeric form, either by chemical cross-linking with bivalent polyethylene glycol (PEG) derivatives or as a genetic fusion with human IgG Fc-fragment, the mutants exhibited a further increase in ligand-binding strength due to the avidity effect, with resident half-times (t(1/2)) on the surface of MHC I-positive cells of many hours. The novel compounds antagonized the interaction of CD8 co-receptor with MHC I in vitro without affecting the peptide-specific binding of T-cell receptors (TCRs). In both cytokine-release assays and cell-killing experiments the engineered receptors inhibited the activation of CD8(+) cytotoxic T lymphocytes (CTLs) in the presence of their target cells, with subnanomolar potency and in a dose-dependent manner. As a selective inhibitor of CD8(+) CTL responses, the engineered high affinity ILT2 receptor presents a new tool for studying the activation mechanism of different subsets of CTLs and could have potential for the development of novel autoimmunity therapies.
Amino Acid Sequence ; Antigens, CD ; chemistry ; genetics ; pharmacology ; Autoimmunity ; Biological Assay ; Cell Line ; Cytotoxicity, Immunologic ; genetics ; immunology ; Dose-Response Relationship, Immunologic ; Humans ; Immunoglobulins ; immunology ; metabolism ; Immunologic Factors ; chemistry ; genetics ; pharmacology ; Kinetics ; Leukocyte Immunoglobulin-like Receptor B1 ; Lymphocyte Activation ; genetics ; immunology ; Major Histocompatibility Complex ; genetics ; immunology ; Molecular Sequence Data ; Molecular Targeted Therapy ; Mutagenesis, Site-Directed ; Peptide Library ; Polyethylene Glycols ; Protein Binding ; genetics ; immunology ; Receptors, Immunologic ; chemistry ; genetics ; Recombinant Fusion Proteins ; genetics ; metabolism ; T-Lymphocytes, Cytotoxic ; immunology ; metabolism

The genomic DNA were extracted from the leaves of Polygonatum roseum (Liliaceae) in Xinjiang and the primers were designed according to conservative sequences of Polygonatum lectins gene. The complete ORF of Polygonatum roseum agglutinin (PRA) gene was amplified as a fragment of 550 bp, which was identical with predicted size. Like most of the plant lectin genes, there was no intron in the PRA gene. The ORF of the gene encoded 159 amino acid residues, in which included a signal sequence of 28 amino acid residues at its N-terminus. The cDNA sequence had 92% identities compared with the published sequence. The amino acid sequence and SWISS-MODEL analysis indicated that the three-dimensional structure of PRA strongly resembled with that of monocot mannose-binding lectins, which comprised with three antiparallel four-stranded beta-sheets arranged as a 12-stranded beta-barrel. The recombinant pGEX4T-1-PRA and pMAL-p2x-PRA prokaryotic expression vectors were constructed to produce GST-PRA and MBP-PRA fusion proteins in E. coli, respectively. SDS-PAGE of the fusion protein demonstrated that the PRA lectin protein migrated at a size of 14 kD. The immunization was performed by intra-muscular injection of pcDNA3-PRA, and the antiserum was detected by ELISA. Western blotting analysis showed the antiserum specifically bound the lectin protein. The establishment of such an expression system might provide materials for further investigation of the properties and functions of PRA proteins. It also laid the basis for plant genetic engineering on its defensive functions to pests and diseases.
Amino Acid Sequence ; Animals ; Base Sequence ; China ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Female ; Mannose-Binding Lectin ; biosynthesis ; genetics ; Mice ; Molecular Sequence Data ; Polygonatum ; chemistry ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology ; Sequence Analysis

BACKGROUND/AIMS: To develop a novel treatment method for hepatitis B virus (HBV) infection, we aimed to make a human monoclonal antibody inhibiting reverse transcriptase (RT) activity of P protein which was important in HBV replication by using phage display technique. Therefore, we analysed the usability of human monoclonal antibody as a protein based gene therapy. METHODS: Reverse transcriptase/polymerase (RT/POL) functional motif of P protein of HBV was cloned in pMAL-c vector and expressed as maltose binding fusion protein form. The RT/POL recombinant protein (pMRT/POL) was purified by amylose resin column. Using human single chain Fv phage antibody library with 1.1x10(10) size, human antibody against pMRT/POL was selected with BIAcore panning. Selected antibody fragments were analyzed for the activity of RT inhibition. Finally, they were analyzed for the affinity with BIAcore and the complementarity determining regions with nucleotide sequencing. RESULTS: pMRT/POL recombinant protein expressed in E. coli showed RT activity, 1microgram of recombinant protein had an activity equivalent to 5 unit of MMLV RT. By BIAcore panning, we could select 3 clones; POL-A5, POL-B8 and POL-B12. Each clone's RT inhibiting activity were 52-82%, affinity against antigen were 8.15x10(-8) M to 1.75x10(-6) M. CONCLUSIONS: Human monoclonal antibodies produced in this study showed low affinity, but efficiently inhibited the activity of RT in vitro. If POL-A5, POL-B8, and POL-B12 can be converted to intracellular antibody form, it can be used for protein-based gene therapy by inhibiting the replication through the neutralization of polymerase protein of HBV.
Antibodies, Monoclonal/biosynthesis/genetics/*pharmacology ; Complementarity Determining Regions/chemistry ; Gene Products, pol/*antagonists & inhibitors/genetics/immunology ; Genetic Vectors ; Hepatitis B virus/enzymology/genetics ; Humans ; Peptide Library ; RNA-Directed DNA Polymerase/genetics/*immunology ; Recombinant Fusion Proteins/biosynthesis/genetics ; Reverse Transcriptase Inhibitors/chemistry/metabolism/*pharmacology

An extracellular matrix protein plays an important role in skin wound healing. In the present study, we engineered a recombinant protein encompassing the 9th and 10th type III domains of fibronectin, and 4th FAS1 domain of beta ig-h3. This recombinant protein, in total, harbors four known-cell adhesion motifs for integrins: Pro-His-Ser-Arg-Asn (PHSRN) and Arg-Gly-Asp (RGD) in 9th and 10th type III domains of fibronectin, respectively, and Glu-Pro-Asp-Ile-Met (EPDIM) and Try-His (YH) in 4th FAS1 domain of big-h3, were designated to tetra-cell adhesion motifs (T-CAM). In vitro studies showed T-CAM supporting adhesion, migration and proliferation of different cell types including keratinocytes and fibroblasts. In an animal model of full-thickness skin wound, T-CAM exhibited excellent wound healing effects, superior to both 4th FAS1 domain of beta ig-h3 or 9th and 10th type III domains of fibronectin. Based on these results, T-CAM can be applied where enhancement of cell adhesion, migration and proliferation are desired, and it could be developed into novel wound healing drug.
Amino Acid Motifs ; Animals ; Cell Adhesion/*drug effects ; Cell Line ; Cell Movement/*drug effects ; Cell Proliferation/*drug effects ; Extracellular Matrix Proteins/chemistry/genetics/pharmacology ; Fibroblasts/cytology/drug effects/physiology ; Fibronectins/chemistry/genetics/*pharmacology ; Humans ; Keratinocytes/cytology/drug effects/physiology ; Mice ; NIH 3T3 Cells ; Rabbits ; Recombinant Fusion Proteins/chemistry/genetics/pharmacology ; Transforming Growth Factor beta/chemistry/genetics/pharmacology ; Wound Healing/*drug effects/physiology

To establish a new high-throughput screening model for the agonist of PPARdelta, PPARdelta gene was obtained by reverse transcriptase-polymerase chain reaction (RT-PCR), and subcloned to pGEM-T Vector for sequencing, then the PPARdelta fragment was excised by restriction enzymes, and inserted into pTARGET Vector to construct expression vector pTARGET-ppARdelta. Insert three copies of PPRE into pGl3-promoter vector to construct expression vector pGl3-PPRE x 3-luc. The vector pTARGET-ppARdelta was transiently cotransfected with pGl3-PPRE x 3-luc into different cell lines to assay the expression levels of luciferase. The PPARdelta agonist screening model was established and optimized. Bezafibrate and linoleic acid can induce the expression of luciferase significantly and in a dose-dependent manner. This method can be used for high throughput screening for the agonist of PPARdelta, which might become lead compounds for new anti-atheroscleriosis or anti-adiposity drugs.
3T3 Cells ; Animals ; Bezafibrate ; pharmacology ; Cell Line ; Dose-Response Relationship, Drug ; Drug Evaluation, Preclinical ; methods ; Genetic Vectors ; chemistry ; genetics ; HeLa Cells ; Humans ; Linoleic Acid ; pharmacology ; Lipids ; chemistry ; Luciferases ; genetics ; metabolism ; Mice ; PPAR delta ; agonists ; genetics ; Recombinant Fusion Proteins ; genetics ; metabolism ; Transfection ; methods