• Journal of Microbiology and Biotechnology
• /
• v.12 no.3
• /
• pp.417-422
• /
• 2002

Gene fusions were constructed by the transcriptional fusion of the dnaK promoter of pseudomonas sp. DJ-12 or E. coli to the lux or luc marker gene. The dnaKp-DJ::luxCDABE bioluminescent fusion in the biosensor using the Pseudomonas sp. DJ-12 dnaK promoter exhibited about 5-fold more extensive response to ethanol than that of dnaKp-EC::luxCDABE. The bioluminescent response of the dnaK-DJ::luc fusion to ethanol was much weaker than those of the other fusions. The biosensor harboring the dnaKp-DJ::luCDABE fusion was examined for its bioluminescence production based on exposure to aromatic compounds, such as biphenyl, 4-chlorobiphenyl (4CB), 4-hydroxybenzoate (4HBA), and catechol. In particular, the bioluminescence produced by the dnaKp-DJ::luxCDABE fusion was most sensitive to 1 mM biphenyl and 4CB when exposed for 80 min, and the responses were also very strong to other aromatics. Therefore, the biosensor bearing the dnaKp-DJ::luxCDABE fusion would appear to be the most useful for the detection of aromatics and other pollutants.

• Journal of Industrial Technology
• /
• v.29 no.A
• /
• pp.161-167
• /
• 2009

The bifunctional Xylanase-Cellulase hybrid protein was constructed by gene fusion. Two genes corresponding to endoxylanase gene (xylS) and endocellulase gene (celA) were amplified by PCR from Bacillus licleniformis NBL420. It was then linked through splicing by overlap extension (SOE) by PCR method. The two resulting fused hybrids, xyl/cel and cel/xyl, which differ by its orientation, were confirmed by its nucleotide sequencings. One of two fusion genes, xyl/cel was successfully expressed into pET22b(+) vector (pxyl/cel) with bifunctional xylanase-cellulase activity. On the contrary, the other cel/xyl fusion protein showed only cellulase activity with much decreased xylanase activity. Enzymatic properties of Xyl/Cel fusion protein were investigated regarding optimum pH, optimum temp, thermostability, and pH stability. It was revealed that Xyl/Cel fusion protein retained the bifunctional xylanase-cellulase activities eventhough two enzymes were connected with each other directly. These informations could be useful for construction of other hybrid proteins as well as increased range of substrate utilization.

• The Journal of Korean Society of Virology
• /
• v.27 no.2
• /
• pp.105-113
• /
• 1997

The truncated $E1_{192-283}$ and $E2_{384-649}$ genes of hepatitis C virus (HCV) linked to the gene for glutathione S-transferase (GST) were constructed and their expressions were analyzed. The $GST-E1_{192-283}$ fusion gene overexpressed the fusion protein in E. coli as a soluble form, while the $GST-E1_{192-383}$ plasmid did not express expected fusion protein. The purified $GST-E1_{192-283}$ fusion protein was efficiently cleaved by thrombin. More than 90% pure, HCV $E1_{192-283}$ protein was obtained by GST-agarose chromatography. The truncated $GST-E2_{384-649}$ fusion gene expressed the fusion protein mainly as an insoluble form, whereas the $GST-E2_{384-740}$ did not express the fusion protein. The truncated $GST-E1_{182-283}$ and $GST-E2_{384-649}$ fusion proteins reacted specifically with an HCV patient serum. In addition, mice immunized with either the purified $E1_{192-283}$ or $GST-E2_{384-649}$ proteins generated specific antibodies to each antigen. The results suggested that hydrophobic carboxyl portions of the E1 and E2 proteins might affect expression levels as well as the solubility of each fusion protein in bacteria. Also, the truncated E1 protein with Tyr-192 to Ser-283 contained antigenic epitope(s) which could be specifically recognized by an HCV patient serum.

• Korean journal of applied entomology
• /
• v.36 no.4
• /
• pp.341-350
• /
• 1997

We have now constructed a novel recombinant baculovirus producing fusion protein with Autographa californica nuclear polyhedrosis virus (AcNPV) polyhedrin and Bacillus thuringiensis(Bt) cryIA(c) crystal protein. The fusion protein expressed by the recombinant baculovirus in insect cells was characterized. The N-terminal of cryIA(c) gene of Bt subsp. kurstaki HD-73 was introduced under the control of polyhedrin gene promoter of AcNPV, by fusion in the front of intact polyhedrin gene or by insertion into the HindIII site in polyhedrin gene. The recombinant baculoviruses were named as BtrusI or BtrusII, respectively. Although single transcript from the fusion protein gene was apparently observed. BtrusI was produced the two proteins, 92 kDa fusion protein and only polyhedrin. In addition, fusion protein produced by BtrusI did not form polyhedra. Interestingly, however, the cells infected with BtrusII did not show a 33 kDa polyhedrin band as a cells infected with BtrusI. Cells infected with BtrusII were only produced fusion protein, but the polyhedra formed by fusion protein was not observed. To determine the insecticidal toxicity of fusion protein, therefore, Sf9 cells infected with BtrusI were inoculated to Bombyx mori larvae. Sf9 cells infected with BtrusI that expressed the fusion protein caused larval mortality although the insecticidal toxicity was low. In conclusion, our results clearly demonstrated that the fusion protein with polyhedrin and Bt cryIA(c) crystal protein have a insecticida toxicity.

• Journal of Microbiology and Biotechnology
• /
• v.20 no.10
• /
• pp.1430-1435
• /
• 2010

Two angiostatic fusion proteins (hAE and hEA), differing in tandem connection manners, were constructed from human angiostatin (hAS) and endostatin (hES) proteins. These fusion proteins were then evaluated for synergistic antiangiogenic properties. The 65 kDa secreted fusion proteins, expressed in Pichia pastoris, were verified by both mass analysis and Western blotting assay. Luciferase reorter gene assay, using a VEGF promoter, revealed that the angiostatin-endostatin fusion protein (hAE), and its corresponding fusion gene delivery on human microvascular endothelial cells (HMEC-1), resulted in a more potent synergistic antiangiogenic effect than the endostatin-angiostatin fusion protein (hEA). These results suggest that the orientation of the fusion genes in hAS and hES might be an important factor in the development of therapeutic proteins.

• BMB Reports
• /
• v.43 no.7
• /
• pp.451-460
• /
• 2010

Starting with the first publication of lacZ gene fusion in 1980, reporter genes have just entered their fourth decade. Initial studies relied on the simple fusion of a promoter or gene with a particular reporter gene of interest. Such constructs were then used to determine the promoter activity under specific conditions or within a given cell or organ. Although this protocol was, and still is, very effective, current research shows a paradigm shift has occurred in the use of reporter systems. With the advent of innovative cloning and synthetic biology techniques and microfluidic/nanodroplet systems, reporter genes and their proteins are now finding themselves used in increasingly intricate and novel applications. For example, researchers have used fluorescent proteins to study biofilm formation and discovered that microchannels develop within the biofilm. Furthermore, there has recently been a "fusion" of art and science; through the construction of genetic circuits and regulatory systems, researchers are using bacteria to "paint" pictures based upon external stimuli. As such, this review will discuss the past and current trends in reporter gene applications as well as some exciting potential applications and models that are being developed based upon these remarkable proteins.

• Genomics & Informatics
• /
• v.19 no.4
• /
• pp.41.1-41.10
• /
• 2021

In addition to mutations and copy number alterations, gene fusions are commonly identified in cancers. In thyroid cancer, fusions of important cancer-related genes have been commonly reported; however, extant panels do not cover all clinically important gene fusions. In this study, we aimed to develop a custom RNA-based sequencing panel to identify the key fusions in thyroid cancer. Our ThyChase panel was designed to detect 87 types of gene fusion. As quality control of RNA sequencing, five housekeeping genes were included in this panel. When we applied this panel for the analysis of fusions containing reference RNA (HD796), three expected fusions (EML4-ALK, CCDC6-RET, and TPM3-NTRK1) were successfully identified. We confirmed the fusion breakpoint sequences of the three fusions from HD796 by Sanger sequencing. Regarding the limit of detection, this panel could detect the target fusions from a tumor sample containing a 1% fusion-positive tumor cellular fraction. Taken together, our ThyChase panel would be useful to identify gene fusions in the clinical field.

• Journal of Microbiology
• /
• v.43 no.1
• /
• pp.44-48
• /
• 2005

In our previous study, the first structural gene (GGTI) encoding ${\gamma}-glutamyl$ transpeptidase was cloned and characterized from the fission yeast Schizosaccharomyces pombe, and its transcription, using the GGTI-lacZ fusion gene, containing the 1,085 bp upstream region from the translational initiation point, was found to be enhanced by sodium nitroprusside and L-buthionine-(S,R)-sulfoximine (BSO). In the present work, regulation of the GGTI gene was further elucidated. Non-fermentable carbon sources, such as acetate and ethanol, markedly enhanced the synthesis of ${beta}-galactosidase$ from the GGTI-lacZ fusion gene. However, its induction by non-fermentable carbon sources appeared to be independent of the presence of the Pap1 protein. Nitrogen starvation also gave rise to induction of GGTI gene expression in a Pap1-independent manner. The three additional fusion plasmids, carrying 754, 421 and 156 bp regions, were constructed. The sequence responsible for the induction by non-fermentable carbon sources and nitrogen starvation was identified to exist within a -421 bp region of the GGTI gene. Taken together, the S. pombe GGTI gene is regulated by non-fermentable carbon sources and nitrogen starvation.

• BMB Reports
• /
• v.38 no.5
• /
• pp.609-618
• /
• 2005

$\gamma$-Glutamyl transpeptidase (GGT, EC 2.3.2.2.) catalyzes the transfer of the $\gamma$-glutamyl moiety from $\gamma$-glutamyl containing ompounds, notably glutathione (GSH), to acceptor amino acids and peptides. A second gene (GGTII) encoding GGT was previously isolated and characterized from the fission yeast Schizosaccharomyces pombe. In the present work, the GGTII-lacZ fusion gene was constructed and used to study the transcriptional regulation of the S. pombe GGTII gene. The synthesis of $\beta$-galactosidase from the GGTII-lacZ fusion gene was significantly enhanced by NO-generating SNP and hydrogen peroxide in the wild type yeast cells. The GGTII mRNA level was increased in the wild-type S. pombe cells treated with SNP. However, the induction by SNP was abolished in the Pap1-negative S. pombe cells, implying that the induction by SNP of GGTII is mediated by Pap1. Fermentable carbon sources, such as glucose (at low concentrations), lactose and sucrose, as a sole carbon source, enhanced the synthesis of $\beta$-galactosidase from the GGTII-lacZ fusion gene in wild type KP1 cells but not in Pap1-negative cells. Glycerol, a non-fermentable carbon source, was also able to induce the synthesis of $\beta$-galactosidase from the fusion gene, but other non-fermentable carbon sources such as acetate and ethanol were not. Transcriptional induction of the GGTII gene by fermentable carbon sources was also confirmed by increased GGTII mRNA levels in the yeast cells grown with them. Nitrogen starvation was also able to induce the synthesis of $\beta$-galactosidase from the GGTII-lacZ fusion gene in a Pap1-dependent manner. On the basis of the results, it is concluded that the S. pombe GGTII gene is regulated by oxidative and metabolic stress.

• Journal of Microbiology and Biotechnology
• /
• v.17 no.5
• /
• pp.728-732
• /
• 2007

In the present article, a novel fusion expression vector for Escherichia coli was developed based on the pTORG plasmid, a derivative of pET32a. This vector, named pT7MT(GenBank Accession No DQ504436), carries a T7 promoter and it drives the downstream gene encoding Metallothionein 2A(MT2A). There are in-framed multiple cloning sites(MCS) downstream of the MT2A gene. A target gene can be cloned into the MCS and fused to the C-terminal of the MT2A gene in a compatible open reading frame(ORF) to achieve fusion expression. The metal-binding capability of MT2A allows the purification of fusion proteins by metal chelating affinity chromatography, known as $Ni^{2+}$-affinity chromatography. Using this expression vector, we successfully got the stable and high-yield expression of MT2A-GST and MT2A-Troponin I fusion proteins. These two proteins were easily purified from the supernatant of cell lysates by one-step $Ni^{2+}$-affinity chromatography. The final yields of MT2A-GST and MT2A-Troponin I were 30mg/l and 28mg/l in LB culture, respectively. Taken together, our data suggest that pT7MT can be applied as a useful expression vector for stable and high-yield production of fusion proteins.