Hemoglobinuria, Paroxysmal ; metabolism ; Humans ; Membrane Proteins ; biosynthesis

OBJECTIVETo study the expression and localization of ATP50 by construction of ATP50-pEYFP-N1 in primary cultured mouse Leydig cells.

METHODSPrimary cultured mouse Leydig cells were confirmed by 3B-HSD staining. ATP50 was cloned into pEYFP-N1 between Bam HI and Eco RI sites. Cell-transfection and living-cell fluorescence imaging microscopy were employed to investigate the sub-cellular localization of YFP-ATP50 in TM3 mouse Leydig cells.

RESULTSATP50 green fluorescent protein was well co-localized with red fluorescence mitochondrion marker-Mitotracker in TM3 mouse Leydig cells.

CONCLUSIONATP50 was expressed in primary cultured mouse Leydig cells. The fluorescent expression vector of ATP50 was constructed successfully and YFP-ATP50 was located in mitochondria in TM3 mouse Leydig cells, which provided a useful clue for further research on the steroidogenesis dysfunction in aging males.

Adenosine Triphosphatases ; biosynthesis ; genetics ; Animals ; Carrier Proteins ; biosynthesis ; genetics ; Cells, Cultured ; Cloning, Molecular ; Genetic Vectors ; Green Fluorescent Proteins ; biosynthesis ; genetics ; Leydig Cells ; metabolism ; Male ; Membrane Proteins ; biosynthesis ; genetics ; Mice ; Mitochondria ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; Transfection

Fuantai-03(FAT-03), isolated from the Dasyatis akajei, has a strong antiangiogenic activity. The recombinant Fuantai-03 (GST/rFAT-03) fusion protein can be obtained with the DNA recombination technology. In this study, expression conditions of GST/rFAT-03 were optimized by response surface experimental design method. The constructed engineering bacteria containing GST/rFAT-03 plasmid was induced by isopropy-beta-D-thiogalactosid (IPTG), the GST affinity column was used for isolation and purification, and then the effects of different culture time, IPTG concentration, induction temperature and induction time on the amount of soluble GST/rFAT-03 fusion protein were compared. The culture time for optimal expression was 6.13 h, IPTG concentration was 0.36 mmol/L, induction temperature was 19.71 degrees C, and induction time was 13.60 h. The amount of soluble GST/rFAT-03 fusion protein was 7.57 mg/L under above mentioned expression conditions. The results also showed that rFAT-03 significantly inhibited angiogenesis in chicken chorioallantoic membrane in a dose-dependent manner. Moreover, the soluble form of the target protein is useful for further work on purification and on studying its biological function.
Angiogenesis Inhibitors ; biosynthesis ; genetics ; Animals ; Chickens ; Chorioallantoic Membrane ; blood supply ; Escherichia coli ; genetics ; metabolism ; Fish Proteins ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Skates (Fish)

This study was aimed to construct a prokaryotic expressing vector of Hap gene from Nontypeable Haemophilus influenzae, and express and identify the fusion proteins of Hap-His in E. coli. The gene encoding protein Hap was amplified from Nontypeable Haemophilus influenzae ATCC49247 chromosomal DNA by PCR, then it was cloned into prokaryotic expression plasmid pET-32a (+). The recombinant plasmid pET-32a(+)-Hap was transformed into E. coli BL21 and expression was induced by Isopropy-beta-D-thiogalatoside(IPTG). The Hap-His fusion protein expressed so was analyzed by SDS-PAGE and Western-blot. The results showed that the recombinant expressive plasmid pET-32a (+)-Hap was constructed successfully, and the recombinant plasmid expressed Hap-His fusion protein with relative molecule mass 176 000 and mainly existed in inclusion body. This fusion protein could combine with anti-His monoclonal antibody specifically through Western blot analysis. Successful expression of Hap-His fusion protein in prokaryotic cell could lay a basis for further study of immunocompetence of Hap protein and development of NTHi vaccine.
Bacterial Outer Membrane Proteins ; biosynthesis ; genetics ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Haemophilus influenzae ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Serine Endopeptidases ; biosynthesis ; genetics

The viral spike protein is the main surface antigen of the coronavirus, and it could be useful in the research of clinical diagnosis, SARS vaccine and the structure biology.According to the analysis of the main antigen of the SARS spike protein, 5 fragments of the whole spike gene were cloned, and ligated to the vector pNMT1. Through electroporation transformantion to TCP1, the recombinant S. pombe strains capable of expressing the 5 fragments were constructed. SDS-PAGE or Western blot analysis of the induced expression products demonstrated that the 5 recombinant proteins were expressed in the fission yeast respectively.
Cloning, Molecular ; Electroporation ; Membrane Glycoproteins ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; SARS Virus ; genetics ; Schizosaccharomyces ; genetics ; metabolism ; Spike Glycoprotein, Coronavirus ; Viral Envelope Proteins ; biosynthesis ; genetics

OBJECTIVETo investigate the role of multidrug resistant protein 2 (MRP2) and glutathione (GSH) cotransport system in hepatic arsenic metabolism in rats.

METHODSThirty healthy Wistar rats were divided randomizedly into five groups. The first group was the control group and the rats in this group were administered with normal saline. In the second, third and fourth group the rats were administered with 4, 10 and 20 mg As(+)3/kg BW of sodium arsenite respectively every other day for two weeks. The fifth group was the benzene-soluble organics (BSO) intervention group and in this group the rats were administered with 2 mmol/kg BW BSO intraperitoneally every day three days before the end of the experiment. The other treatment was the same as in other groups. All rats were sacrificed two weeks after the treatments. Arsenic contents in bile, liver and blood were detected by atomic absorption spectroscopy (AAS), and the expression of MRP2 in the membrane of hepatocyte was determined by Western-blot analysis.

RESULTSThe level of total arsenic (including organic arsenic and inorganic arsenic) in bile, liver and blood in all three different dose groups was higher than those in the control groups (P < 0.05). Arsenic levels of bile and liver were increased with intragastric arsenic dose. Blood arsenic levels were not significantly different in three different dose groups. Expression of hepatic MRP2 was increased with intragastric arsenic concentration. A positive correlation between biliary arsenic concentration and MRP2 levels was found in liver (r = 0.986, P < 0.05). For the rats pretreated with BSO, the biliary arsenic was significantly higher than that in the control group but lower than that in the high dose group; the liver and blood arsenic was higher than that in the control group and in the high dose group. Expression of MRP2 pretreated with BSO was decreased.

CONCLUSIONSodium arsenite can induce expression of MRP2 and the up-regulation of MRP2 may play an important role in the bile secretion of arsenite and its metabolites. The function of MRP2 for transportation of arsenic and its metabolites is associated with the intracellular GSH level. BSO inhibits the synthesis of GSH, which weakens the function of the MRP2-GSH cotransport system and makes the liver arsenic increased.

Animals ; Arsenic ; pharmacokinetics ; Arsenic Poisoning ; metabolism ; Bile ; metabolism ; Female ; Glutathione ; biosynthesis ; Liver ; metabolism ; Male ; Membrane Transport Proteins ; biosynthesis ; Multidrug Resistance-Associated Proteins ; biosynthesis ; Random Allocation ; Rats ; Up-Regulation

Omics docking study for polygenic inheritance tumors has become an important strategy in oncology research. This review focuses on the conceptions and technologies of omics, and puts forward the central contents and omics docking for polygenic inheritance tumor to reveal the role of molecular changes at different stages of polygenic inheritance tumor at multidisciplinary and multilayer level. It is a new strategy to explore the mechanism of tumor carcinogenesis, and to regulate the network, key molecules, and drug target by combined biology effects.
Carrier Proteins ; biosynthesis ; genetics ; Genomics ; methods ; Glycoproteins ; biosynthesis ; genetics ; Humans ; Membrane Proteins ; biosynthesis ; genetics ; Multifactorial Inheritance ; genetics ; Neoplasms ; genetics ; metabolism ; Phosphoproteins ; biosynthesis ; genetics ; Proteomics ; methods ; Tumor Suppressor Proteins ; biosynthesis ; genetics

The expression of a soluble protein on cell surface is often desirable for study of a functional protein, wide application of a protein or investigation of protein-protein interaction. The expression of a soluble protein on the surface of a cell is often achieved by genetically linking a protein to the extra-cellular fragment of a transmembrane partner. In this study, the myc epitope was linked with N terminal of transmembrane proteins either A2TM or deltaLNGFR amplified by overlapping PCR. The plasmids expressing fusion protein were transfected into 293FT cells and the expression of target proteins was evaluated by fluorescent microscope, flow cytometry and Western blotting. The results of flow cytometry revealed that both A2TM and deltaLNGFR were expressed on the cell surface, but A2TM could only be detected with high copy number. Western blotting showed that the expression level of deltaLNGFR was very high and protein was heavily glycosylated, by contrast the expression of A2TM was hardly detected. The results indicate that glycosylated deltaLNGFR is a good candidate partner for the expression of a soluble protein on the cell surface.
Apoptosis Regulatory Proteins ; biosynthesis ; genetics ; Cell Membrane ; metabolism ; Genes, MHC Class II ; genetics ; HLA-A2 Antigen ; biosynthesis ; genetics ; Humans ; Membrane Fusion Proteins ; genetics ; metabolism ; Membrane Proteins ; genetics ; metabolism ; Proto-Oncogene Proteins c-myc ; metabolism

OBJECTIVETo identify and clone the gene named pfstom gene which encoding the protein belonging to band 7 family and to do primary research on its function.

METHODSBased on the finished data in international public malaria database, coding sequence of pfstom cDNA was obtained by RT-PCR from FCC1/HN. Its phylogenetic profiles and the homogeny were analyzed by some softwares. After Prokaryotic expression, C terminal of Pfstom protein was expressed by Pet30a system. Recombinant Pfstom protein was used to immol/Lunize rabbit and then serum was harvested and the IgG was purified for Western blot.

RESULTSThe coding sequence of pfstom is 1,125 bp which encoding 374 amino acids with C-terminal fragment being homogenous to stomatin-like protein which belongs to band 7 family. Phylogenetic profiles analysis revealed its homogeny to stomatin. Western blot showed its stage-specific expression in trophozoite.

CONCLUSIONPfstom belongs to band-7 family. It was expressed specifically in trophozoite in erythrocyte stage of plasmodium falciparum. It was not expressed in ring stage. And it is membrane-related protein. All these results provided the foundation for further research on pfstom.

Amino Acid Sequence ; Animals ; Blood Proteins ; biosynthesis ; genetics ; immunology ; DNA, Complementary ; genetics ; Membrane Proteins ; biosynthesis ; genetics ; immunology ; Molecular Sequence Data ; Phylogeny ; Plasmodium falciparum ; chemistry ; genetics ; pathogenicity ; Protozoan Proteins ; biosynthesis ; genetics ; immunology ; Rabbits ; Recombinant Proteins ; biosynthesis ; genetics

OBJECTIVE: To explore the down-expression mechanism of MYETS1 gene in multiple myeloma cell lines ARH-77 or KM3, and express MYETS1 gene in prokaryotic express system. METHODS: The region of chromosome 13q14.3 in ARH-77 and KM3 was detected by FISH. MYETS1 gene was amplified by RT-PCR and cloned into prokaryotic expression vector pGEX-4T. RESULTS: Positive consequence was acquired in 13q14.3 where MYETS1 located by FISH in ARH- 77 and KM3 cell lines. Bioinformatics indicated highly sequence homology between MYETS1 and LECT1, but excluded the homology of open reading frame between MYETS1 and that of LECT1 by RT-PCR. Myets1 protein was expressed and harvested successfully. CONCLUSION The region of chromosome 13q14.3 ,where MYETS1 gene located, was not defected in ARH-77 and KM3 cell lines. Down-expression of MYETS1 might be regulated by other mechanisms in multiple myeloma cell lines.
Cell Line, Tumor ; Chromosomes, Human, Pair 13 ; genetics ; Gene Deletion ; Genetic Vectors ; genetics ; Humans ; Intercellular Signaling Peptides and Proteins ; biosynthesis ; genetics ; Membrane Proteins ; biosynthesis ; genetics ; Multiple Myeloma ; genetics ; metabolism ; pathology ; Neoplasm Proteins ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics