To detect the expression of telomerase subunits (human telomerase reverse transcriptase, human telomerase associated protein 1 and human telomerase RNA) in gastric cancer and to examine the role that different telomerase subunits play in the gastric carcinogenesis, reverse transcription-polymerase chain reaction (RT-PCR) was used to detect telomerase subunits messenger RNA in 24 samples of gastric cancer and corresponding non-cancerous tissue. The results showed that the positive rate of hTERT mRNA from gastric cancer and corresponding non-cancerous tissues was 100% and 25%, respectively. The former was significantly higher than the latter (chi2 = 26.4, P < 0.01). The positive rate of hTEP1 mRNA from gastric cancer and corresponding non-cancerous tissues was 100% and 91.7%, respectively and no significant difference was found between them (chi2 = 2.1, P > 0.05). The positive rates of hTR for gastric cancer and corresponding non-cancerous tissues were both 100% and no significant difference existed between them. It is concluded that in contrast to hTEP1 and hTR, the up-regulation of hTERT mRNA expression may play a more important role in the development of gastric cancer.
Carrier Proteins/biosynthesis ; Carrier Proteins/genetics ; RNA/biosynthesis ; RNA/genetics ; RNA, Messenger/biosynthesis ; RNA, Messenger/genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Stomach Neoplasms/*metabolism ; Telomerase/*biosynthesis ; Telomerase/genetics

Antibodies, Bacterial ; blood ; Antigens, Bacterial ; biosynthesis ; Bacterial Proteins ; biosynthesis ; Carrier Proteins ; biosynthesis ; Gastritis ; microbiology ; Helicobacter Infections ; immunology ; Helicobacter pylori ; immunology ; isolation & purification ; Humans ; Recombinant Proteins ; biosynthesis

OBJECTIVE: To analyze the expression and location of coding protein of UBAP1 gene and to understand the relationship between the expression pattern of the protein and cell carcinogenesis. METHODS: Bioinformatics was used to analyze the protein character to provide an available clue of subsequent research. The codon frame cDNA was amplified by PCR, and subcloned into enhance green fluorescence protein (EGFP) of pEGFP-C2. The recombinant plasmid was transfected into HNE1 cells. The expression of coding protein was observed by fluorescence microscopy. RESULTS: The expressed GFP-fusion protein generated striking green fluorescence in the cytoplasm in HNE1 cells. EGFP/UBAP1 was expressed and existed mainly in the nuclear, especially accumulated on the nuclear envelope. CONCLUSION The expression difference in HNE1 might be related to the carcinogenesis of NPC.
Base Sequence ; Carrier Proteins ; Green Fluorescent Proteins ; biosynthesis ; genetics ; Humans ; Molecular Sequence Data ; Nasopharyngeal Neoplasms ; metabolism ; Neoplasm Proteins ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Transfection

OBJECTIVETo express SPAG4L, a novel human testis gene in E. coli and purify it's fusion protein.

METHODSThe fragment encoding SPAG4L126-379 was amplified by RT-PCR and the PCR products were cloned into PUCm-T vectors. After digestion by EcoR I and Hind III, the fragment was subcloned into PQE-30, a prokaryotic expression vector with 6×His tag. The recombinant plasmid PQE-30-SPAG4L was sequenced and transformed into E.coli M15. The expression of his-tagged fusion protein was induced by IPTG. The fusion protein was identified by Western blotting and purified using Ni-NTA magnetic agarose beads.

RESULTSThe recombinant plasmid PQE-30-SPAG4L was constructed successfully and expressed in E.coli M15. The fusion protein SPAG4Lwith 6×his-tag was confirmed by Western blotting. The micro-scale purification system of 6×His-tagged SPAG4Lprotein was established and purified fusion protein was obtained.

CONCLUSIONThe recombinant plasmid PQE-30-SPAG4L can be expressed in vitro and used for studying the biological function of SPAG4L in spermatogenesis.

Carrier Proteins ; biosynthesis ; genetics ; isolation & purification ; Escherichia coli ; genetics ; metabolism ; Humans ; Male ; Plasmids ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification

D-amino acid oxidase (DAAO) is one of important industrial enzymes. To increase the solubility and activity of the TvDAAO from Trignoposis variabilis expressed in recombinant Escherichia coli (E. coli), a maltose binding protein (MBP) and Vitreoscilla hemoglobin (VHb) was introduced to fuse with N-terminal of the TvDAAO, respectively. Fusion protein of MBP-TvDAAO was constitutively expressed in JM105/pMKC-DAAO and inductively expressed in JM105/pMKL-DAAO. With respect to the control strain of BL21 (DE3)/pET-DAAO without MBP fusion, the constitutive fusion expression obtained 28% of soluble protein with 3.7 folds of solubility improvement. As for the inductive fusion expression, corresponding results changed to 17% and 1.8 folds, respectively. However, the DAAO activity significantly decreased in the MBP-fusing expression. Fusion protein of VHb-TvDAAO was constructed and inductively expressed in BL21 (DE3)/pET-VDAAO. Its DAAO activity highly reached 3.24 u/mL in flask culture, about 90% increase in contrast to the control without VHb.
Bacterial Proteins ; biosynthesis ; genetics ; Carrier Proteins ; biosynthesis ; genetics ; D-Amino-Acid Oxidase ; biosynthesis ; genetics ; Escherichia coli ; genetics ; metabolism ; Maltose-Binding Proteins ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Truncated Hemoglobins ; biosynthesis ; genetics ; Yeasts ; enzymology ; genetics

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

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

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

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

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