In order to research the biologic activity of osteoblast-stimulating factor 1 (OSF-1), the pPIC9K/osf-1 yeast expression vector was constructed to express and purify OSF-1. Firstly, the osf-1 gene sequence was obtained by artificial synthesis and cloned into Pichia pastoris expression vector pPIC9K to generate pPIC9K/osf-1. The recombinant plasmid was linearized by Sac I and transformed into P. pastoris GS115 by electroporation. Recombinant P. pastoris GS115/ pPIC9K/osf-1 was screened by MD and G418-YPD plates and further identified by PCR. The positive P. pastoris was induced with 1% methanol at 25 degrees C for 96 h. The target protein was analyzed by SDS-PAGE showing a special band about 18 kDa. The target protein was successfully purified from the supernatant of the broth using ion exchange chromatography of SP-Sephadex C-50. The purity of target protein was above 98%. Western blotting appeared a good antigenicity of the purified protein. Bioassay results show that the recombinant protein OSF-1 can promote the differentiation and proliferation of osteoblasts MC3T3-E1. We successfully expressed OSF-1 by recombinant P. pastoris for further development of anti-osteoporosis of research and industrial production of OSF-1.
Blotting, Western ; Carrier Proteins ; biosynthesis ; Chromatography, Affinity ; Cytokines ; biosynthesis ; Electrophoresis, Polyacrylamide Gel ; Electroporation ; Pichia ; metabolism ; Polymerase Chain Reaction ; Recombinant Proteins ; biosynthesis

Fatty acid synthase (FAS) catalyses the reaction between acetyl-CoA and malonyl-CoA to produce fatty acids. It is one of the most important enzyme in lipid biosynthesis. FAS of the oleaginous yeast Rhodosporidium toruloides has two acyl carrier protein (ACP) domains and a distinct subunit composition compared with FASs of other species. As ACP is a protein cofactor crucial for fatty acid chain elongation, more ACPs in the FAS may facilitate the reaction. To study the biochemical and structural properties of this novel FAS from R. toruloides, plasmids were constructed and transformed into Escherichia coli BL21 (DE3). The strain ZWE06 harboring plasmids pET22b-FAS1 and pET24b-FAS2 could co-overexpress the two subunits. The recombinant FAS was purified by sequentially using ammonium sulphate precipitation, sucrose density gradient centrifugation and anion exchange chromatography. The specific activity of the recombinant FAS was 548 mU/mg. The purified complex would be used to study enzyme kinetics and protein structure of FAS, and heterogeneous expression and purification will facilitate revealing the mechanism of this novel FAS with double ACPs.
Acyl Carrier Protein ; Basidiomycota ; enzymology ; Chromatography ; Escherichia coli ; metabolism ; Fatty Acid Synthases ; biosynthesis ; genetics ; Fatty Acids ; biosynthesis ; Plasmids ; Recombinant Proteins ; biosynthesis ; genetics

The aim of this study is to establish stable transfected cell lines which could produce SPAG4L protein labeled with Myc and His tags in vitro. The open reading frame (ORF) of human SPAG4L was amplified by PCR and the fragments were cloned into eukaryotic expression vector pcDNA3.1/myc-His(-)A. The recombined plasmids of pcDNA3.1/myc-His(-)A/SPAG4L were verified by sequencing and digestion with enzymes. Then, the recombined plasmids were introduced into HeLa cells and screened by G418. Western blotting was performed to detect the expression of SPAG4L and its tags in stable transfected cell lines. SPAG4L and its tags were expressed in the stable cell lines transfected with pcDNA3.1/myc-His(-)A/SPAG4L, but not in the control group. Further study showed that SPAG4L colocalized with the endoplasmic reticulum(ER) marker PDI by immunofluorescence. The stable transfected cell lines established in this study will provide a powerful tool for further studies such as co-immunoprecipitation and pull-down.
Carrier Proteins ; biosynthesis ; genetics ; Cloning, Molecular ; Female ; Genetic Vectors ; genetics ; HeLa Cells ; Histidine ; genetics ; Humans ; Male ; Proto-Oncogene Proteins c-myc ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification ; Transfection

We evaluated the effectiveness of rhamnogalacturonan II (RG-II)-stimulated bone marrow-derived dendritic cells (BMDCs) vaccination on the induction of antitumor immunity in a mouse lymphoma model using EG7-lymphoma cells expressing ovalbumin (OVA). BMDCs treated with RG-II had an activated phenotype. RG-II induced interleukin (IL)-12, IL-1beta, tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) production during dendritic cell (DC) maturation. BMDCs stimulated with RG-II facilitate the proliferation of CD8+ T cells. Using BMDCs from the mice deficient in Toll-like receptors (TLRs), we revealed that RG-II activity is dependent on TLR4. RG-II showed a preventive effect of immunization with OVA-pulsed BMDCs against EG7 lymphoma. These results suggested that RG-II expedites the DC-based immune response through the TLR4 signaling pathway.
Acute-Phase Proteins/metabolism ; Adaptor Proteins, Vesicular Transport/metabolism ; Animals ; Antigens, CD14/metabolism ; Bone Marrow Cells/cytology/drug effects ; CD8-Positive T-Lymphocytes/*immunology ; Carrier Proteins/metabolism ; Cell Differentiation/drug effects ; Cell Nucleus/drug effects/metabolism ; Cell Proliferation/drug effects ; Cytokines/biosynthesis ; Dendritic Cells/cytology/drug effects/enzymology/*immunology ; Enzyme Activation/drug effects ; Lymphocyte Activation/*drug effects ; Membrane Glycoproteins/metabolism ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Mitogen-Activated Protein Kinases/metabolism ; Myeloid Differentiation Factor 88/metabolism ; NF-kappa B/metabolism ; Neoplasms/immunology/*pathology ; Pectins/*pharmacology ; Phenotype ; Protein Transport/drug effects ; Receptors, Chemokine/metabolism ; Signal Transduction/drug effects ; T-Lymphocytes, Cytotoxic/cytology/drug effects ; Toll-Like Receptor 4/*agonists/metabolism

The fungus Trichophyton schoenleinii (T. schoenleinii) is the causative agent of Trichophytosis and Tinea favosa of the scalp in certain regions of Eurasia and Africa. Human innate immune system plays an important role in combating with various pathogens including fungi. The inflammasome is one of the most critical arms of host innate immunity, which is a protein complex controlling maturation of IL-1β. To clarify whether T. schoenleinii is able to activate the inflammasome, we analyzed human monocytic cell line THP-1 for IL-1β production upon infection with T. schoenleinii strain isolated from Tinea favosa patients, and rapid IL-1β secretion from THP-1 cells was observed. Moreover, applying competitive inhibitors and gene specific silencing with shRNA, we found that T. schoenleinii induced IL-1β secretion, ASC pyroptosome formation as well as caspase-1 activation were all dependent on NLRP3. Cathepsin B activity, ROS production and K⁺ efflux were required for the inflammasome activation by T. schoenleinii. Our data thus reveal that the NLRP3 inflammasome plays an important role in host defense against T. schoenleinii, and suggest that manipulating NLRP3 signaling can be a novel approach for control of diseases caused by T. schoenleinii infection.
Animals ; Bone Marrow Cells ; cytology ; Carrier Proteins ; metabolism ; Caspase 1 ; metabolism ; Cell Line ; Dendritic Cells ; cytology ; metabolism ; microbiology ; Enzyme Activation ; Hot Temperature ; Humans ; Inflammasomes ; metabolism ; Interleukin-1beta ; biosynthesis ; metabolism ; Lysosomes ; metabolism ; Mice ; Monocytes ; cytology ; metabolism ; microbiology ; NLR Family, Pyrin Domain-Containing 3 Protein ; Potassium ; metabolism ; Reactive Oxygen Species ; metabolism ; Signal Transduction ; Trichophyton ; physiology

The adapter protein Lamellipodin (Lpd) plays an important role in cell migration. In particular, Lpd mediates lamellipodia formation by regulating actin dynamics via interacting with Ena/VASP proteins. Its RA-PH tandem domain configuration suggests that like its paralog RIAM, Lpd may also mediate particular Ras GTPase signaling. We determined the crystal structures of the Lpd RA-PH domains alone and with an N-terminal coiled-coil region (cc-RA-PH). These structures reveal that apart from the anticipated coiled-coil interaction, Lpd may also oligomerize through a second intermolecular contact site. We then validated both oligomerization interfaces in solution by mutagenesis. A fluorescence-polarization study demonstrated that Lpd binds phosphoinositol with low affinity. Based on our crystallographic and biochemical data, we propose that Lpd and RIAM serve diverse functions: Lpd plays a predominant role in regulating actin polymerization, and its function in mediating Ras GTPase signaling is largely suppressed compared to RIAM.
Actins ; metabolism ; Amino Acid Sequence ; Binding Sites ; Carrier Proteins ; chemistry ; genetics ; metabolism ; Crystallography, X-Ray ; Humans ; Membrane Proteins ; chemistry ; genetics ; metabolism ; Molecular Sequence Data ; Mutagenesis ; Phosphatidylinositols ; metabolism ; Polymerization ; Protein Binding ; Protein Structure, Tertiary ; Recombinant Proteins ; biosynthesis ; chemistry ; genetics ; Signal Transduction ; ras Proteins ; metabolism

OBJECTIVETo express granzyme B-vascular endothelial growth factor (VEGF) receptor-binding peptide (GrB-VRB) fusion protein in Bifidobacteria longum (B. longum) and investigate the effects of this fusion protein on the proliferation and apoptosis of cells expressing VEGF receptor II, the kinase domain receptor (KDR).

METHODSThe recombinant expression vectors pBBADx-VRB, pBBADx-GrB and pBBADx-GrB-VRB were separately transformed into B. longum cells by electroporation. The expressed products were identified by enzyme-linked immunosorbent assay and Western blotting, and their effects on KDR-positive cells were analyzed using proliferation assay and TUNEL assay.

RESULTSThe expressed products were detected in both the supernatant and cellular fractions of B. longum cells. The recombinant GrB-VRB fusion protein reacted with such KDR-positive cells as human umbilical vein endothelial cells (HUVEC) and mouse colon cancer cell line CT-26, and caused obvious cell proliferation inhibition, cytotoxicity and cell apoptosis in these cells.

CONCLUSIONThe recombinant GrB-VRB fusion protein secreted by the engineered B. longum cells can induce KDR-positive cell death as the result of GrB-induced cell apoptosis following the cell recognition by VRB.

Animals ; Apoptosis ; Bifidobacterium ; metabolism ; Carrier Proteins ; Cell Line, Tumor ; Cell Proliferation ; Granzymes ; metabolism ; Human Umbilical Vein Endothelial Cells ; Humans ; Mice ; Receptors, Vascular Endothelial Growth Factor ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; Vascular Endothelial Growth Factor Receptor-2 ; metabolism

OBJECTIVETo construct the eukaryotic expression vector of HBEBP1 gene and express HBEBP1 recombinant protein in yeast.

METHODSPCR was performed to amplify the gene of HBEBP1 from the cDNA template origining from HepG2, and the gene was cloned into pGEM-T vector. After sequencing, the correct DNA fragment was cut from pGEM-T-HBEBP1 and inserted into yeast expression plasmid pGBKT7. The reconstructed plasmid pGBKT7-HBEBP1 was transformed into yeast cell AH109 and screened on the synthetic dropout nutrient medium (SD/-Trp/Kana). The yeast protein was isolated and analyzed with SDS-PAGE and Western Blot.

RESULTSThe eukaryotic expressive vector was constructed successfully. The results of Western Blot showed HBEBP1 protein was existed within yeast cells and the molecular weight of it was about 33 x 10(3).

CONCLUSIONSThe successful expression of HBEBP1 protein in yeast cells lay the foundation for studying biological function of HBEBP1.

Blotting, Western ; Carrier Proteins ; genetics ; Hepatitis B e Antigens ; metabolism ; Plasmids ; Recombinant Proteins ; biosynthesis ; Saccharomyces cerevisiae ; genetics

Biotin is an important micronutrient that serves as an essential enzyme cofactor. Bacteria obtain biotin either through de novo synthesis or by active uptake from exogenous sources. Mycobacteria are unusual amongst bacteria in that their primary source of biotin is through de novo synthesis. Here we review the importance of biotin biosynthesis in the lifecycle of Mycobacteria. Genetic screens designed to identify key metabolic processes have highlighted a role for the biotin biosynthesis in bacilli growth, infection and survival during the latency phase. These studies help to establish the biotin biosynthetic pathway as a potential drug target for new anti-tuberculosis agents.
Biotin ; biosynthesis ; Carbon-Carbon Ligases ; metabolism ; Carrier Proteins ; metabolism ; Cell Membrane ; metabolism ; Coenzymes ; metabolism ; Fatty Acids ; biosynthesis ; Genes, Bacterial ; Genome, Bacterial ; Metabolic Networks and Pathways ; Molecular Structure ; Mycobacterium Infections ; microbiology ; Mycobacterium tuberculosis ; genetics ; metabolism ; pathogenicity ; physiology ; Virulence

Cyanovirin-N (CVN) is an 11 kDa anti-HIV protein originally isolated from extracts of a cyanobacterium, Nostoc ellipsosporum. The protein binds with high affinity to the viral envelope glycoprotein gp120 and irreversibly inactivates diverse HIV strains. A fusion gene consisting of cvn, sumo and 6xHis tag was synthesized by PCR according to the codon bias of Escherichia coli. The fusion protein is expressed in the cytoplasm of E. coli in a soluble form and up to 28% of the total protein. The recombinant CVN was purified to homogeneity by 2 rounds of Ni-NTA affinity chromatography and one round of SUMO protease cleavage. Bioactivity assay demonstrated that SUMO-CVN and CVN bound to gp120 with nanomolar concentration. In addition, CVN showed potent anti-HSV-1 and anti-HIV-1 activities in in vitro cellular assays. Therefore, the 6xHis SUMO fusion expression and purification system provides a better approach for large scale production of CVN for further microbicide development.
Antiviral Agents ; isolation & purification ; metabolism ; pharmacology ; Bacterial Proteins ; biosynthesis ; genetics ; pharmacology ; Carrier Proteins ; biosynthesis ; genetics ; pharmacology ; Escherichia coli ; genetics ; metabolism ; HIV-1 ; drug effects ; Herpesvirus 1, Human ; drug effects ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification ; pharmacology