Acrosin ; immunology ; isolation & purification ; Humans ; Male ; Membrane Proteins ; immunology ; isolation & purification ; Protein Binding ; Spermatozoa ; metabolism ; Zona Pellucida ; metabolism

OBJECTIVETo screen the molecules binding dengue II virus expressed in Aedes albopictus C6/36 cells and characterize their biological functions.

METHODSAedes albopictus C6/36 cells were infected with dengue II virus, and the virus were collected and purified. The total and membrane proteins of C6/36 cells were extracted and analyzed using 12% SDS-polyacrylamide gel (PAGE). After electrophoresis, the proteins were transferred to a nitrocellulose membrane, and virus overlay protein-binding assay (VOPBA) was carried out using an anti-dengue virus 1-4 monoclonal antibody.

RESULTSTwo specific bands of 67 000 and 30 000 occurred after VOPBA of the proteins from the cells incubated with the virus, while the negative control group did not show these specific bands.

CONCLUSIONTwo putative dengue virus receptor molecules of 67 000 and 30000 have been obtained from C6/36 cells using VOPBA, and their functional identification is in progress.

Aedes ; cytology ; virology ; Animals ; Cells, Cultured ; Dengue Virus ; isolation & purification ; Membrane Proteins ; Receptors, Virus ; isolation & purification ; metabolism ; Virus Attachment

Animals ; Hepatocytes ; metabolism ; Male ; Membrane Proteins ; isolation & purification ; Rats ; Rats, Sprague-Dawley

Two Heptad repeat motifs (HR1 and HR2) from paramyxoviruses F protein could form thermostable heterodimers containing high alpha-helix while virus infected host cell. Following that the viral membrane and the host cell membrane were juxtaposed, which leads to membrane fusion. Mumps virus (MuV) is a member of the genus Rubulavirus in the family of Paramyxoviridae. MuV could use similar infection mechanism as well as other paramyxoviruses. In this study the HR1 and HR2 regions of MuV F protein were predicted by a computer program and expressed in E. coli with the GST fusion expression system. The GST fusion or GST-removed proteins were purified with Gluthathion Sepharose 4B Column. GST pull-down experiment suggested the interaction of HR1 and HR2 peptides, and analysis of gel filtration showed two peptides could form multimer, which indicates that the HR regions of MuV F protein may play an important role in virus fusion.
Membrane Fusion ; genetics ; Mumps virus ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; chemistry ; genetics ; isolation & purification ; Repetitive Sequences, Amino Acid ; Viral Fusion Proteins ; biosynthesis ; genetics ; isolation & purification

Pectobacterium carotovorum subsp. carotovorum is one of the world's top ten plant pathogens, mainly infecting cruciferous economic crops and ornamental flowers. In this study, an antibacterial gene cpxP (Gene ID: 29704421) was cloned from the genome of Pectobacterium carotovorum subsp. carotovorum, and constructed on the prokaryotic expression plasmid pET-15b, and the recombinant plasmid was transformed into Escherichia coli BL21 (DE3), then stability and bacteriostatic experiments of the purified CpxP protein were performed. The final concentration of IPTG was 1 mmol/L, obtaining high-efficiency exogenous expression of the CpxP protein. There was no other protein after purification, and the destined protein exhibited good thermal stability and pH stability. The antibacterial test results showed that the inhibition rate of the CpxP protein on carrot slice was 44.89% while the inhibition rate on potato slice was 59.41%. To further explain its antibacterial mechanism, studying the spatial structure of this protein can provide new ideas for the control of soft rot and new protein pesticide targets.
Anti-Bacterial Agents ; pharmacology ; Bacteria ; drug effects ; Bacterial Proteins ; isolation & purification ; pharmacology ; Escherichia coli ; genetics ; Membrane Proteins ; isolation & purification ; pharmacology ; Pectobacterium carotovorum ; genetics ; metabolism ; Plasmids ; genetics

OBJECTIVETo purify IbeA-binding protein from intestinal epithelial Caco-2 cells.

METHODSRecombinant IbeA was purified, and 1, 5, and 10 microg/ml His-IbeA and bovine serum albumin (control) were preincubated with confluent Caco-2 monolayer for 30 min at 4degrees celsius;. Gentamicin protection assay was used to test the invasion of E. coli K1 pathogenic isolate E44 in Caco-2 cells. The binding proteins were purified from Caco-2 by IbeA-Cu(2+) sepharose affinity chromatography, and validated by Far-Western blotting. The N-terminal amino acid sequence of the binding protein was determined using Edman assay.

RESULTSE44 invasion in Caco-2 cells was blocked by the recombinant IbeA in a dose-dependent manner. Two binding bands were obtained with His pull-down, and the binding specificity was demonstrated by Far-Western blotting. The N-terminal amino acid sequence of IBP200 was MASITKLP with an isoelectric point of about 5.0.

CONCLUSIONTwo novel Caco-2 proteins interacting with IbeA of E. coli have been purified and identified.

Caco-2 Cells ; Epithelial Cells ; cytology ; metabolism ; microbiology ; Escherichia coli Proteins ; genetics ; isolation & purification ; metabolism ; Humans ; Intestines ; cytology ; metabolism ; Membrane Proteins ; genetics ; isolation & purification ; metabolism ; Recombinant Proteins ; genetics ; isolation & purification

To construct a recombinant plasmid Pet23a-M, the gene encoding severe acute respiratory syndrome (SARS) coronavirus membrane protein was amplified by RT-PCR and cloned into the expression plasmid Pet23a. Results of restriction endonuclease analysis, PCR detection and DNA sequencing analysis revealed that the cloned DNA sequence was the same as that reported. The recombinants were transformed into Escherichia coli (E. Coli) BL21 (DE3) and induced by Isopropyl-beta-D-thiogalactopyranoside (IPTG). The expression of 27 kD (1 kD=0.992 1 ku) protein was detected by SDS-PAGE and pured by metal chelated chromatography. Results of Western-blot showed that this expressed protein could react with antibodies in sera of SARS patients during convalescence. This provided the basis for the further study on SARS virus vaccine and diagnostic agents.
Cloning, Molecular ; Escherichia coli/genetics ; Escherichia coli/metabolism ; Membrane Proteins/*biosynthesis ; Membrane Proteins/genetics ; Membrane Proteins/isolation & purification ; Plasmids/biosynthesis ; Plasmids/genetics ; Recombinant Proteins/biosynthesis ; Recombinant Proteins/genetics ; Recombinant Proteins/isolation & purification ; Reverse Transcriptase Polymerase Chain Reaction ; SARS Virus/chemistry ; SARS Virus/*genetics ; Viral Vaccines/biosynthesis

OBJECTIVETo explore the feasibility of membrane protein-based two-dimensional electrophoresis (2-DE) in the investigations of chondrocyte-related diseases and its complementarity with total protein-based 2-DE.

METHODSKnee cartilage samples were obtained to isolate the chondrocytes with type II collagenase/hyaluronidase digestion. The membrane proteins and total proteins were extracted and loaded separately onto PH3.0-10.0 non-linear gel strip for 2-DE analysis. The qualities of membrane protein-based 2-DE gels were evaluated, and the difference between the distribution profiles of the membrane protein spots and that of the total protein were observed and their complementarities were evaluated.

RESULTSMembrane protein-based 2-DE generated high-quality gel images, and on each gel 412.3±13.5 protein spots were identified. These spots were distributed in the region of isoelectric point pH 5.0-9.0. In the gel images generated by the total proteins, 564.3±5.9 protein spots were identified in each image, and the spots were distributed in the region of isoelectric point pH 3.0-7.0.

CONCLUSIONMembrane protein-based 2-DE of the chondrocytes can generate high-quality gel images, and the isoelectric distribution of the protein spots is complementary to that of total protein, which provides valuable information for chondrocyte-related diseases.

Adult ; Chondrocytes ; chemistry ; Electrophoresis, Gel, Two-Dimensional ; methods ; Female ; Humans ; Isoelectric Point ; Male ; Membrane Proteins ; isolation & purification ; Middle Aged

OBJECTIVETo investigate the feasibility of obtaining of a highly pure protein of human endothelial overexpressed lipopolysaccharide-associated factor 1 (EOLA1) with metal chelation chromatography.

METHODSInclusion bodies of the E. coli transformed with EOLA1 gene were extracted and washed with BugBuster Protein Extraction Reagent. The primary purified products were purified by His. Bind Resin Chromatography under denaturing condition and dialyzed for renaturation, and then were analyzed with SDS-PAGE, Western blotting and peptide mass fingerprinting (PMF).

RESULTSEOLA1 was mainly expressed in E. coli as insoluble inclusion bodies. The protein content in the primary extracted inclusion bodies accounted for over 75%, and it accounted for more than 90% after chromatography and renaturation. It was indicated by PMF that the targeted protein peptide overlaid many of designed protein peptide.

CONCLUSIONThe method of EOLA1 protein purification and renaturation was convenient and efficient, and by this method sufficient amount of highly pure EOLA1 protein could be obtained for the preparation of EOLA1 monoclonal antibody and for the study of its gene function.

Antibodies, Monoclonal ; Chromatography, Affinity ; Electrophoresis, Polyacrylamide Gel ; Endothelial Cells ; metabolism ; Humans ; Immunoblotting ; Membrane Proteins ; genetics ; isolation & purification ; metabolism

OBJECTIVETo construct the expression plasmid of S2 extracellular domain (S2ED) of SARS-coronavirus (SARS- Cov) spike protein (S protein) and enhanced green fluorescent protein (EGFP) to obtain the fusion protein expressed in prokaryotic cells.

METHODSS2ED based on bioinformatics prediction and EGFP sequence were amplified by PCR and inserted into pET-14b plasmid. The recombinant protein His-S2ED-EGFP was expressed in E. coli by IPTG induction. After purification by Ni-NTA agarose beads, the soluble fractions of the fusion protein were collected and identified by SDS-PAGE and Western blotting. The fusion of S2ED with Hela cell membranes was observed with fluorescent microscope.

RESULTSThe pET-14b-S2ED-EGFP plasmid was correctly constructed and highly expressed in BL21 (DE3). When incubated with Hela cells, the purified protein could not internalize through membrane fusion.

CONCLUSIONSThe expression plasmid containing S2ED of SARS-Cov S protein and EGFP sequence is constructed successfully. Although the recombinant protein obtained has not shown the expected fusion effect with Hela cell membrane, this work may enrich the understanding of the process of membrane fusion mediated by S2 protein and lay the foundation for future study of targeting cell transport system based on cell-specific binding peptide.

Escherichia coli ; genetics ; metabolism ; Green Fluorescent Proteins ; biosynthesis ; genetics ; metabolism ; HeLa Cells ; Humans ; Membrane Fusion ; drug effects ; Membrane Fusion Proteins ; biosynthesis ; isolation & purification ; Membrane Glycoproteins ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification ; SARS Virus ; genetics ; Spike Glycoprotein, Coronavirus ; Viral Envelope Proteins ; biosynthesis ; genetics