• Journal of Microbiology and Biotechnology
• /
• v.16 no.6
• /
• pp.933-938
• /
• 2006

Decaprenyl diphosphate synthase (DPS) is the key enzyme for the production of coenzyme $Q_{10}$ ($CoQ_{10}$). A dps gene from Sinorhizobium meliioti KCCM 11232 (IFO 14782) was isolated by PCR and then cloned in Escherichia coli. DNA sequencing analysis revealed an open reading frame of 1,017 bp encoding a 338-amino-acid protein. The protein was identical at the 98% level to the putative octaprenyl diphosphate synthase (IspB) of S. meliloti 1021. The deduced amino acid sequence included the DDxxD domains conserved in the majority of the prenyl diphosphate synthases. Heterologous expression in E. coli BL21 (DE3) was carried out, and the $CoQ_{10}$ produced was then analyzed by HPLC. E. coli BL21 (DE3) harboring the dps gene from S. melioti produced CoQ$_{10}$ in addition to endogenous coenzyme Q$_8$ (CoQ$_8$), whereas wild-type E. coli BL21 (DE3) host did not have the ability of producing CoQ$_{10}$. The results suggest that the putative dps from S. meliloti KCTC 2353 encoded the DPS.

• Journal of Microbiology and Biotechnology
• /
• v.25 no.7
• /
• pp.1007-1014
• /
• 2015

Plectasin, the first defensin extracted from a fungus (the saprophytic ascomycete Pseudoplectania nigrella), is attractive as a prospective antimicrobial agent. The purpose of this study was to establish a bacterium-based production system and evaluate the antimicrobial activity of the resulting plectasin. A gene encoding plectasin, with the codon preference of Escherichia coli, was optimized based on its amino acid sequence, synthesized using genesplicing with overlap extension PCR, and inserted into the expression vector pGEX-4T-1. The fusion protein was expressed in the soluble fraction of E. coli and purified using glutathione Stransferase affinity chromatography. Plectasin was cleaved from the fusion protein with thrombin and purified by ultrafiltration. The purified plectasin showed strong, concentrationdependent antimicrobial activity against gram-positive bacteria, including antibiotic-resistant bacteria, especially penicillin-resistant Enterococcus faecium. This antimicrobial activity was equal to chemically synthesized plectasin and was maintained over a wide range of pH and temperatures. This soluble recombinant expression system in E. coli is effective for producing plectasin at a relatively lower cost, and higher purity and efficiency than prior systems, and might provide a foundation for developing a large-scale production system. Overall, plectasin shows potential as a novel, high-performance, and safe antibiotic for the treatment of refractory diseases caused by drug-resistant bacterial strains.

• Journal of Microbiology and Biotechnology
• /
• v.21 no.8
• /
• pp.830-837
• /
• 2011

A genomic DNA fragment encoding a putative maltooligosyltrehalose trehalohydrolase (NfMTH) for trehalose biosynthesis was cloned by the degenerate primer- PCR from cyanobacterium Nostoc flagelliforme. The ORF of NfMTH is 1,848 bp in length and encodes 615 amino acid residues, constituting a 70 kDa protein. The deduced amino acid sequence of NfMTH contains 4 regions highly conserved for MTHs. By expression of NfMTH in E. coli, the function of this protein was demonstrated, where the recombinant protein catalyzed the hydrolysis of maltooligosyl trehalose to trehalose. The expressions of MTH and maltooligosyltrehalose synthase in the filaments of N. flagelliforme were upregulated significantly under dehydration stress, NaCl stress, and high temperature-drought stress. The accumulations of both trehalose and sucrose in the filaments of N. flagelliforme were also improved significantly under the above stresses. Furthermore, trehalose accumulated in smaller quantities than sucrose did when under NaCl stress, but accumulated in higher quantities than sucrose did when under temperature-drought stress, indicating that both trehalose and sucrose were involved in N. flagelliforme adapted to stresses and different strategies conducted in response to various stress conditions.

• Journal of Microbiology and Biotechnology
• /
• v.19 no.2
• /
• pp.194-203
• /
• 2009

$HpaG_{Xooc}$, from rice pathogenic bacterium Xanthomonas oryzae pv. oryzicola, is a member of the harpin group of proteins, eliciting hypersensitive cell death in non-host plants, inducing disease and insect resistance in plants, and enhancing plant growth. To express and secret the $HpaG_{Xooc}$ protein in Bacillus subtilis, we constructed a recombinant expression vector pM43HF with stronger promoter P43 and signal peptide element nprB. The SDS-PAGE and Western blot analysis demonstrated the expression of the protein $HpaG_{Xooc}$ in B. subtilis. The ELISA analysis determined the optimum condition for $HpaG_{Xooc}$ expression in B. subtilis WBHF. The biological function analysis indicated that the protein $HpaG_{Xooc}$ from B. subtilis WBHF elicits hypersensitive response(HR) and enhances the growth of tobacco. The results of RT-PCR analysis revealed that $HpaG_{Xooc}$ induces expression of the pathogenesis-related genes PR-1a and PR-1b in plant defense response.

• Korean Journal of Microbiology
• /
• v.52 no.4
• /
• pp.495-499
• /
• 2016

In order to identify the biochemical characteristics of LuxG, the luxG gene from bioluminescence bacteria of Photobacterium leiognathi ATCC 25521 was isolated by PCR-Amplification and inserted into pQE30 vector containing the T5 promoter and 6X His-tag system. The resulting recombinant plasmid was transformed into Escherichia coli to over-express the luxG gene and purify the gene product. The purified LuxG protein demonstrated ferric iron reductase activity and the kinetic parameters of $K_m$ and $V_{max}$ for FMN as well as the NADPH substrates of ferric iron reductase were determined, respectively.

• Animal cells and systems
• /
• v.11 no.2
• /
• pp.175-182
• /
• 2007

The lysozyme II gene of cabbage butterfly Artogeia rapae was cloned from fat body of the larvae injected with E. coli and its nucleotide sequence was determined by the RACE-PCR. It has an open reading frame of 414 bp nucleotides corresponding to 138 amino acids including a signal sequence of 18 amino acids. The estimated molecular weight and the isoelectric point of the lysozyme II without the signal peptide were 13,649.38 Da and 9.11, respectively. The A. rapae lysozyme II (ARL II) showed the highest identity (81%) in the amino acid sequence to Manduca sexta lysozyme among other lepidopteran species. The two catalytic residues ($Glu^{32}$ and $Asp^{50}$) and the eight Cys residue motifs, which are highly conserved among other c-type lysozymes in invertebrates and vertebrates, are also completely conserved. A phylogenetic analysis based on amino acid sequences indicated that the ARL II was more closely related to M. sexta, Hyphantria cunea, Heliothis virescens, and Trichoplusia ni lysozymes. The ARL II gene was expressed in Spodoptera frugiperda 21 insect cells and the recombinant ARL II (rARL II) was purified from cell-conditioned media by cation exchange column chromatography and reverse phase FPLC. The purified rARL II was able to form a clear zone in lysoplate assay against Micrococcus luteus. The lytic activity was estimated to be 511.41 U/mg, 1.53 times higher than that of the chicken lysozyme. The optimum temperature for the lytic activity of the rARL II was $50^{\circ}C$, the temperature dependency of the absolute lytic activity of rARL II was higher than that of the chicken lysozyme at low temperatures under $65^{\circ}C$.

• KSBB Journal
• /
• v.16 no.6
• /
• pp.562-567
• /
• 2001

Aspergillus niger LK harboring the enantioselective epoxide hydrolase (EHase) activity was isolated, and enantioselectivity of EHase was tested for various racemic aromatic epoxides. The gene encoding epoxide hydrolase was cloned from cDNA library generated by reverse transcriptase-polymerase chain reaction of the isolated total mRNA. Sequence analysis showed that the cloned gene encodes 398 amino acids with a deduced molecular mass of 44.5 kDa. Database comparison of the amino acid sequence reveals that it is similar to fungal EHase, whereas the sequence identity with bacterial EHase is very low. Recombinant expression of the cloned EHase in Escherichia coli BL21 yielded an active EHases, which can offer a potential biocatalyst for the production of chiral epoxides.

• Asian Pacific Journal of Cancer Prevention
• /
• v.15 no.4
• /
• pp.1531-1537
• /
• 2014

Runt-related transcription factor 3 (RUNX3) is a tumor suppressor gene whose reduced expression may play an important role in the development and progression of esophageal squamous cell cancer (ESCC). The aim of this study was to investigate the clinical relevance of RUNX3 in ESCC patients and effects of overexpression on biological behaviour of Eca109 cells in vitro and in vivo. Immunohistochemistry was performed to detect the clinical relevance of RUNX3 and lymph node metastasis in 80 ESCC tissues and 40 non-cancerous tissues using the SP method. RT-PCR and Western blotting were applied to assess the RUNX3 level and verify the Eca109 cell line with stable overexpression. Localization of RUNX3 proteins was performed by cell immunofluorescence. CCK-8 and Scrape motility assays were used to determine proliferation and migration and the TUNEL assay to analyze cell apoptosis. Invasive potential was assessed in cell transwell invasion experiments. In nude mice, tumorigenesis in vivo was determined. Results showed decreased expression of RUNX3 in esophageal tissue to be significantly related to lymph node metastasis (LNM) (P<0.01). In addition, construction of a recombinant lentiviral vector and transfection into the human ESCC cell line Eca109 demonstrated that overexpression could inhibit cell proliferation, migration and invasion, and induce apoptosis. The in vivo experiments in mice showed tumorigenicity and invasiveness to be significantly reduced. Taken together, our studies indicate that underexpression of RUNX3 in human ESCC tissue is significantly correlated with progression. Restoration of RUNX3 expression significantly inhibits ESCC cells proliferation, migration, invasion and tumorigenesis.

• The Journal of Korean Society of Virology
• /
• v.27 no.2
• /
• pp.115-128
• /
• 1997

Three cDNA fragments located within NS5 region of HCV were synthesized by RT using viral RNA extracted from blood sample of Korean patient as a template. The cDNAs were amplified by PCR, cloned into the T-vector, and the nucleotide sequences were determined. Comparative analysis of the nucleotide and amino acid sequence of NS5 cDNAs showed that it is closely related with HCV type 1b. The cloned NS5 cDNA showed 91-94% homology at the nucleotide sequence level and 96-98% homology at the amino acid sequence level with several strains of the HCV type 1b. The NS5 cDNAs were subcloned into E. coli expression vectors to construct pRSETA5-1, pTHAN5-1, pRSETC5-2, pRSETBB1, pRESTCB1 and pRSETB-H3. Expression of the NS5 proteins was achieved by inducing the promoter with isopropyl-thio-${\beta}$-D-galactoside (IPTG) and confirmed by SDS-polyacrylamide gel electrophoresis. The NS5 proteins were immunoreactive against sera from Korean hepatitis C patients in Western blot analysis. Among the recombinant NS5 proteins, pRSETAS-1 plasmid derived protein, coded from aa2022 to aa2521 of HCV polyprotein, showed the strongest immunoreactivity against sera from Korean hepatitis C patients in immunoblot analysis. These results suggest that NS5 proteins would be useful as an antigen for detection of antibody against HCV in the blood samples.

• Asian Pacific Journal of Cancer Prevention
• /
• v.13 no.5
• /
• pp.1845-1849
• /
• 2012

Objective: Tissue factor (TF) is expressed abnormally in certain types of tumor cells, closely related to invasion and metastasis. The aim of this study was to construct a human gastric cancer cell line SGC7901 stably-transfected with human TF, and observe effects on oxaliplatin-dependent inhibition of invasion and the apoptosis induction. Methods: The target gene TF was obtained from human placenta by nested PCR and introduced into the human gastric cell line SGC7901 through transfection mediated by lipofectamine. Stably-transfected cells were screened using G418. Examples successfully transfected with TF-pcDNA3 recombinant (experimental group), and empty vector pcDNA3 (control group) were incubated with oxaliplatin. Transwell chambers were used to show change in invasive ability. Caspase-3 activity was detected using a colorimetric method and annexin-V/PI double-staining was applied to detect apoptosis. Results: We generated the human gastric cancer cell line SGC7901/TF successfully, expressing TF stably and efficiently. Compared with the control group, invasion increased, whereas caspase-3 activity and apoptosis rate were decreased in the experimental group. Conclusion: TF can enhance the invasive capacity of gastric cancer cells in vitro. Its increased expression may reduce invasion inhibition and apoptosis-inducing effects of oxaliplatin and therefore may warrant targeting for improved chemotherapy.