• Journal of Microbiology and Biotechnology
• /
• v.16 no.9
• /
• pp.1362-1368
• /
• 2006

A new constitutive episomal expression vector, pGAPZ-E, was constructed and used for initial screening of eukaryotic target gene expression in Pichia pastoris. Two reporter genes such as beta-galactosidase gene and GFPuv gene were overexpressed in P. pastoris. The expression level of the episomal pGAPZ-E strain was higher than that of the integrated form when the beta-galactosidase gene was used as the reporter gene in P. pastoris X33. The avoiding of both the integration procedure and an induction step simplified the overall screening process for eukaryotic target gene expression in P. pastoris. Nine human protein targets from the Core 50, family of Northeast Structural Genomics Consortium (http://www.nesg.org), which were intractable when expressed in E. coli, were subjected to rapid screening for soluble expression in P. pastoris. HR547, HR919, and HR1697 human proteins, which had previously been found to express poorly or to be insoluble in E. coli, expressed in soluble form in P. pastoris. Therefore, the new episomal GAP promoter vector provides a convenient and alternative system for high-throughput screening of eukaryotic protein expression in P. pastoris.

• BMB Reports
• /
• v.29 no.4
• /
• pp.359-364
• /
• 1996

By both in vitro hydroxylamine mutagenesis of the wild type 3-phosphoglycerate kinase gene (PGK) promoter DNA and insertion of the leu2-d gene, we have created yeast expression vectors potentially useful for production of eukaryotic genes in yeast. The guanine (G) to adenine (A) change at the -3 position from the ATG start codon of the PGK promoter-based vector rendered a 6~7 times elevated expression of the adjacent eukaryotic gene, and insertion of the leu2-d gene in the vector containing the mutated PGK promoter further enhanced the expression of the gene. When expression of the AIDS virus HIV1-gagP17 gene in a lacZ fusion form was examined with this new vector, a 15 times higher level of expression than that from the original PGK promoter was observed. Northern and Southern analysis showed that this elevated expression is due to the production of a high copy number of mRNA by leu2-d gene functioning and by efficient translation of the produced mRNA. Thus, the vector that contained the A at the -3 position from the ATG start codon in the promoter region and the leu2-d gene shows increased expression capability and will be potentially useful for production of eukaryotic genes in yeast.

• Asian Pacific Journal of Cancer Prevention
• /
• v.14 no.1
• /
• pp.507-510
• /
• 2013

Background: More and more research indicate that the immediately early response gene 3 (IER3) is involved inmany biological provesses, such as apoptosis and immunoreaction, as well as viral infection, tumorigenesis and tumour progression. Methods: Here we describe the construction of an eukaryotic expression vector containing IER3 gene and its expression in A549 cells as assessed through fluorescence microscopyand Western-blotting. Results: Fluorescence detection displayed that GFP in cytoplasm was high during 48 and 72 hours post-transfection. In addition, Western blotting showed significant increase in IER3 gene expression in the transfected cells compared with controls. Conclusion: The recombinate plasmid expression vector was constructed successfully, which may provide a basis for further exploration of function of IER3 in lung cancer.

• IMMUNE NETWORK
• /
• v.3 no.1
• /
• pp.23-28
• /
• 2003

Background: Intracellular antibody specific to hepatitis B virus X protein (HBx) might be useful for studying the role of HBx in hepatocellular carcinogenesis and HBV replication. Methods: With variable region genes for H7 monoclonal anti-HBx Ab, we constructed a vector for bacterial expression of single chain Ab (scFv) and a vector for eukaryotic cell expression of it. The expression of H7 scFv and its binding activity against HBx was examined by immunoblotting and immunofluorescence microscopy. Results: H7 scFv expressed in bacterial cells retained reactivity to HBx. We demonstrated its intracytoplasmic expression in CosM6 eukaryotic cells. Conclusion: This is the first study showing the expression of intracellular anti-HBx Ab in eukaryotic cells. H7 scFv may be a good tool to study the function of HBx in HBV infection.

• The Journal of Korean Society of Virology
• /
• v.28 no.2
• /
• pp.139-146
• /
• 1998

Recombinant polioviruses have been developed by many research groups for use as vaccine vector because poliovirus induces mucosal immunity as well as humoral immunity through oral uptake. We assessed the potential use of poliovirus as a T-cell epitope carrier. Recombinant poliovirus V129 5L was constructed to have a substituted T-helper epitope from the core protein of Hepatitis B virus at neutralization antigenic site 1 on its VP1 capsid protein. The recombinant virus replicated less efficiently than type 1 poliovirus Mahoney strain. The V129 5L formed a little smaller plaques than the Mahoney strain and showed some 1.25 log unit lower titer at the peak in the one-step growth kinetics though it had similar growth profile to that of the Mahoney strain. Since V129 5L recombinant virus was genetically stable even after 24 successive passages in HeLa cells, the antigenic site 1 on VP1 capsid protein was confirmed for its ability of carrying T cell epitope. The genetic stability of V129 5L also indicated that recombinant poliovirus can be successfully utilized for the development of the multivalent vaccines.

• Journal of Life Science
• /
• v.16 no.3 s.76
• /
• pp.365-368
• /
• 2006

Yeast mitochondrial transformation has been confirmed by cell death and CFP expression (CDF: cell death factor gene). Expression vector containing CDF and CFP driven by one TPI (Triose-phosphate isomerase) promoter (called artificial operon type) was bombarded to Yeast. Interestingly, yeast cells were progressively deformed into unusual shapes and lysed inner cytoplasm resulting in ell death after all after bombarding with expression vector (CDC and GFP). Since there is no report about more than one gene expression simultaneously in a single mitochondria, this report is very important to novel type of eukaryotic gene expression. Successful yeast cell transformation in this report implies possible eukaryotic mitochodrial transformation including plants and animals and moreover two or more gene expression which can be excellent applicable protocols to pharmaceutical field including antibody production.

• Asian Pacific Journal of Cancer Prevention
• /
• v.14 no.5
• /
• pp.3001-3003
• /
• 2013

Objective: Spleen tyrosine kinase (Syk) is closely related to tumor invasion and metastasis, and has been shown to have potential inhibitory effects in tumors. In this study, we constructed a eukaryotic expression vector for Syk and analyzed its effects on invasive ability of the A549 non-small cell lung cancer cell line in vitro. Methods: A fragment of Syk was obtained by RT-PCR from human lung cancer cells and cloned into the expression vector pLNCXSyk. After restriction endonuclease digestion, PCR and DNA sequencing confirmation, the recombinant Syk expression plasmid was transfected into A549 human lung cancer cells using lipofectamine protocols. After selection, the cells stably expressed Syk. Detection of Syk expression of the cells by RT-PCR, and invasive ability were examined. Results: The eukaryotic expression plamid pLNCXSyk was constructed and expressed stably in the A549 human lung cancer cells. The RT-PCR results showed that Syk mRNA expression was upregulated significantly (P<0.05). Lower invasion through a basal membrane were apparent after transfection (P<0.05). Conclusions: A eukaryotic expression plasmid to cause Syk expression in lung cancer cells can obviously inhibit their invasive ability in vitro.

• Korean Journal of Plant Resources
• /
• v.21 no.4
• /
• pp.249-253
• /
• 2008

Since there is no report about more than one gene expression simultaneously in a single mitochondria, this report is very important to novel type of eukaryotic gene expression. In this study, investigated whether mitochondrial expressed gene and GFP that expression in mitochondria of plant expressed to mitochondria. Expression vector (pBin) containing AtBI-1 (mitochondrial expressed gene) and GFP driven by 35S promoter was bombarded by gene gun to leaves and cotyledon of tobacco. Regenerated shoot confirmed expression of AtBI-1 in mitochondria by GFP expression, PCR, and Southern analysis. Successful mitochondria of plant cell transformation in this report implies possible eukaryotic mitochondrial transformation including plants and animals, and moreover two or more gene expression which can be excellent applicable protocols to pharmaceutical field including antibody production.

• Journal of Microbiology and Biotechnology
• /
• v.12 no.2
• /
• pp.306-311
• /
• 2002

O-linked N-acetylglucosamine (O-GlcNAc) is an abundant posttranslationally modified compound in eukaryotic cells. Human O-GlcNAc transferase (OGT) was produced as a maltose binding protein (MBP) fusion protein, which showed significant catalytic activity to modify recombinant Sp1, transcription factor. To facilitate the production of O-GlcNAc modified proteins, instead of using the tedious in vitro glycosylation reaction or expression in eukaryotic cells, a MBP-fusion OGT expression vector (pACYC184-MBPOGT) was constructed using pACYC184 plasmid, which could coexist with general prokaryotic expression vectors containing ColE1 origin. By cotransforming pACYC184-MBPOGT and pGEX-2T vectors into Escherichia coli BL21, intracellular O- GlcNAcylated proteins could be obtained by a simple purification procedure. It is expected that this may be a useful tool for production of O-GlcNAc modified proteins.

• Asian Pacific Journal of Cancer Prevention
• /
• v.14 no.9
• /
• pp.5467-5472
• /
• 2013

Objective: To construct short hairpin RNA (shRNA) eukaryotic expression vectors targeting Livin and Survivin genes, and to explore the impact of co-transfection of Livin and Survivin shRNA expression vectors on the biological behavior of HepG2 cells. Methods: shRNA eukaryotic expression vectors pSD11-Livin and pSD11-Survivin were designed and constructed then transfected into HepG2 cells separately or in combination. mRNA and protein expression in transfected cells was assessed by quantitative fluorescence PCR and Western blotting, respectively. Cell proliferation was measured by MTT assay and cell apoptosis by TUNEL assay. Results: The Livin and Survivin shRNA eukaryotic expression vectors were successfully constructed and transfected into HepG2 cells. The relative mRNA expression levels of Livin and Survivin in HepG2 cells co-transfected with pSD11-Livin and pSD11-Survivin were $0.12{\pm}0.02$ and $0.33{\pm}0.13$, respectively, which was significantly lower than levels in cells transfected with either pSD11-Livin or pSD11-Survivin (P<0.05). The relative protein expression levels of Livin and Survivin in the co-transfected cells were also significantly decreased compared to single-transfection (P<0.05). The inhibition rate of cell growth in the co-transfection group was higher than that in the single-transfection groups at 48 h, 60 h, or 72 h after transfection (P<0.01). The apoptotic rate increased to the greatest extent in the co-transfection group relative to any other group (P<0.05). Conclusions: Co-transfection with pSD11-Livin and pSD11-Survivin was more efficient than transfection with either vector alone in reducing the mRNA and protein expression of Livin and Survivin genes in HepG2 cells. Co-transfection also inhibited the proliferation of transfected cells more than the other groups, and induced cellular apoptosis more effectively.