• 미생물학회지
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• 제26권2호
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• pp.101-105
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• 1988

6M-MuLV mutants containing deldtions around the putative packaging signal were constructed by using recombinant DNA technique and transfected into NIH/3T3 cell. 2 of 6 mutants can not be packaged into virions even in the presence of the wild type helper virus. The boundary between the packagible and the non-packagible genome is located around Pvu I site, 421 nucleotide downstream from the 5' end of M-MuLV genome. 10 base pair inverted repeat sequence (GAGUCCAAAA) which can make stem structure around Pvu Isite could be the putative packaging signal.

• 생명과학회지
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• 제11권1호
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• pp.43-47
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• 2001

A mutant tryptophan synthase $\alpha$-subunit, where Pro28 was replaced with Leu, tends to be expressed in recombinant E. coli. CD and fluorescence spectra of this protein indicate some changes in secondary and tertiary structure. Wild type protein was more or less affected by {TEX}$Ca^{2+}${/TEX} ion in regards of the fluorescent properties of its native, unfolded and intermediate forms, but the mutant protein was not at all. The dramatic structural changes may be related to the aggregation of this mutant protein.

• Journal of Photoscience
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• 제2권1호
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• pp.27-29
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• 1995

Site directed mutagenesis has been introduced to determine active site(s) and molecular structure of E. coli RecA protein. Recombinant DNAs were constructed by point mutation of Tyr-264 to Phe which assumed active site for binding and hydrolysis of ATP. RecA proteins were purified from recombinants containing wild type and mutant genes and analyzed for ATPase activity assay. Result suggests that Tyr-264 is involved in ATP binding rather than ATP hydrolysis.

• 한국미생물학회:학술대회논문집
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• 한국미생물학회 2002년도 추계학술대회
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• pp.121-124
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• 2002

This work was initiated to develope a recombinant oral poliovaccine (OPV), which is highly advanced in safety (minimizing VAPP) by introducing Type 2,3 poliovirus epitopes into our RPS-Vax system. We have introduced several potential vaccine epitopes of poliovirus Type 2, and 3 into RPS-Vax system, resulting in production of recombinant polioviruses. Any of these chimeric viruses, however, were not detected for their foreign gene expression by serotype-specific mouse antiserum. We have designed several folding units to stabilize the introduced vaccine protein and attached short epitope-concatamer or epitope-multimer to them, followed by production of chimeric viruses. Only those who have an HIV-1 Tat-mediated folding unit were nicely detected for the introduced foreign proteins by anti-Tat antiserum and type-specific peptide-induced antisera. Nevertheless, introduced epitopes were not detected in Western blot experiment with each serotype-specific antiserum. None of the mice inoculated with these chimeric viruses showed preventative immunity when challenged with Lansing and Leon wildtype 2 and 3 poliovirus, and the antiserum did not show neutralizing capacity in vitro. Conformational epitope covering B/C loop region of type 2 and 3 were newly designed by computer modeling, and introduced into the RPS-Vax vector system, followed by production of chimeric viruses. Introduced epitope regions were nicely detected by anti-Tag23 mAb or peptide antibody, but still not detected by poliovirus antiserum. Nevertheless, neutralizing antibody was detected in the Tg-PVR mice even when inoculated once with these chimeric viruses. Also, the immunized mice showed perfect preventative immunity against the wild Type poliovirus Lancing or Leon. When boosted appropriately, those chimeric virus-inoculated Tg-PVR mice produced equivalent amounts of neutralizing antibody to those in Sabin 2/3-immunized mice. These data strongly suggest that our recombinant poliovirus (RPS-PV2 and RPS-PV3) can be used as a safe and effective rec-OPV instead of any preexisting poliovaccine.

• Journal of Genetic Medicine
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• 제3권1호
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• pp.33-37
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• 1999

Recombinant adenovirus vector systems with strong promoters have been used to achieve high level production of recombinant protein. However, this overexpression system cause some problems such as disturbance of cell physiology and increment of cellular toxicity. Here, we showed a tetracycline-regulated adenovirus expression vector system. Our results showed that the expression level of transgene(p-53) was high and easily regulated by tetracycline. In addition, the maximal gene expression level of the tetracycline-controlled gene expression system was higher than that of the wild type CMV promoter system. Therefore, tetracycline-regulated adenoviral vector system could be applicable for regulatory high-level expression of toxic gene. Also, this system will be useful for functional studies and gene therapy.

• Journal of Microbiology and Biotechnology
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• 제28권9호
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• pp.1482-1492
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• 2018

Fusarium oxysporum trypsin (FOT) is a fungal serine protease similar to mammal trypsin. The FOT could be successfully expressed in Pichiapastoris by engineering the natural propeptide APQEIPN. In this study, we constructed two recombinant enzymes with engineered amino acid sequences added to the N-terminus of FOT and expressed in P. pastoris. The N-terminal residues had various effects on the structural and functional properties of trypsin. The FOT, and the recombinants TE (with peptide YVEF) and TS (with peptide YV) displayed the same optimum temperature ($40^{\circ}C$) and pH (8.0). However, the combinants TE and TS showed significantly increased thermal stability at $40^{\circ}C$ and $50^{\circ}C$. Moreover, the combinants TE and TS also showed enhanced tolerance of alkaline pH conditions. Compared with those of wild-type FOT, the intramolecular hydrogen bonds and the cation ${\pi}$-interactions of the recombinants TE and TS were significantly increased. The recombinants TE and TS also had significantly increased catalytic efficiencies (referring to the specificity constant, $k_{cat}/K_m$), 1.75-fold and 1.23-fold than wild-type FOT. In silico modeling analysis uncovered that the introduction of the peptides YVEF and YV resulted in shorter distances between the substrate binding pocket (D174, G198, and G208) and catalytic triad (His42, Asp102, and Ser180), which would improve the electron transfer rate and catalytic efficiency. In addition, N-terminal residues modification described here may be a useful approach for improving the catalytic efficiencies and characteristics of other target enzymes.

• Parasites, Hosts and Diseases
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• 제53권4호
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• pp.385-394
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• 2015

Leishmaniasis is a worldwide uncontrolled parasitic disease due to the lack of effective drug and vaccine. To speed up effective drug development, we need powerful methods to rapidly assess drug effectiveness against the intracellular form of Leishmania in high throughput assays. Reporter gene technology has proven to be an excellent tool for drug screening in vitro. The effects of reporter proteins on parasite infectivity should be identified both in vitro and in vivo. In this research, we initially compared the infectivity rate of recombinant Leishmania major expressing stably enhanced green fluorescent protein (EGFP) alone or EGFP-luciferase (EGFP-LUC) with the wild-type strain. Next, we evaluated the sensitivity of these parasites to amphotericin B (AmB) as a standard drug in 2 parasitic phases, promastigote and amastigote. This comparison was made by MTT and nitric oxide (NO) assay and by quantifying the specific signals derived from reporter genes like EGFP intensity and luciferase activity. To study the amastigote form, both B10R and THP-1 macrophage cell lines were infected in the stationary phase and were exposed to AmB at different time points. Our results clearly revealed that the 3 parasite lines had similar in vitro infectivity rates with comparable parasite-induced levels of NO following interferon-${\gamma}$/lipopolysaccharide induction. Based on our results we proposed the more reporter gene, the faster and more sensitive evaluation of the drug efficiency.

• KSBB Journal
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• 제13권2호
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• pp.214-219
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• 1998

Polymerase chain reaction(PCR) was conducted to obtain the gene encoding glucose oxidase(GOD) from Aspergillus niger(ATCC 2110) and the DNA sequence determined was coincided with published GOD sequence from A. niger. Recombinant transforming vector containing GOD and hygromycin B(hyg.B) resistant gene(hph) was constructed and used for further transformation of A. niger ATCC 2110. Selectivity of hyg.B against A. niger differed depending on which media were used i.e., nutrient-rich media such as potato dextrose agar(PDA) and complete medium(CM) showed only 50% growth inhibition at 400 $\mu$m ml$^-1$ of hyg.B while the minimal media inhibited mycelial growth completely at 200 $\mu$m ml$^-1$ of hyg.B. Twenty to sixty putative transformants were isolated from the hyg.B-containing minimal top agar, transferred successively onto alternating selective and nonselective media for a mitotic stability of hyg.B resistance and, then, single-spored. Among the stable transformants, the transformant(GOD1-6) grown by flask culture showed the considerable increase of extracellular GOD activity, which was estimated to the degree of 50% - 100% comparing to that of wild type. Transformation of tGOD1-6 was resulted from integration of the vectors into heterologous as well as homologous regions of the A. niger genome. Southern blot analysis revealed that there were two independent integrations of vector into fungal genome and one into the GOD gene due to homologous recombination. In addition, GOD activity and sodium gluconate production when tGOD1-6 was fed-batch fermented were enhanced 11 fold and 2.25 fold, respectively, compared to that of the wild type.

• 한국동물위생학회지
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• 제28권4호
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• pp.393-405
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• 2005

In prion pathogenesis, the structural conversion of the cellular prion protein $(PrP^c)$ to its abnormal isomer $(PrP^{Sc})$ is believed to be a major event. The susceptibility or resistance to natural sheep scrapie is associated with polymorphisms of host PrP gene (PRNP) at amino acid residues 136, to a lesser extent 154. The 112 residue in ovine PrP displays a natural polymorphism, Methionine to Threonine, which has not been thoroughly investigated. However the cell-free conversion assay showed that ARQ with Thr112 $(T_{112}ARQ)^{1)}$ presents lower convertibility to $PrP^{Sc}$than wild type ARQ $(M_{112}ARQ)$ [1] In this study we generated ovine recombinant PrPs of 112 allelic variants by metal chelate affinity chromatography and cation exchange chromatography. The final purity of the ovine PrP ARQ was more than $95\%$. These variants showed similar immunoreactivity against anti-PrP monoclonal antibodies in Western blot and ELISA. The refolded $M_{112}ARQ$ and $M_{112}ARQ$ presented the secondary structural content to similar extent via CD spectroscopy analysis. The inherited structural features of $M_{112}ARQ$ and $M_{112}ARQ$ under the different biophysical conditions are in the middle of investigation.

• Journal of Microbiology and Biotechnology
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• 제33권3호
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• pp.310-318
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• 2023

Microalgae are attracting much attention as promising, eco-friendly producers of bioenergy due to their fast growth, absorption of carbon dioxide from the atmosphere, and production capacity in wastewater and salt water. However, microalgae can only accumulate large quantities of lipid in abiotic stress, which reduces productivity by decreasing cell growth. In this study, the strategy was investigated to increase cell viability and lipid production by overexpressing S-adenosylmethionine (SAM) synthetase (SAMS) in the microalga Chlamydomonas reinhardtii. SAM is a substance that plays an important role in various intracellular biochemical reactions, such as cell proliferation and stress response, and the overexpression of SAMS could allow cells to ithstand the abiotic stress and increase productivity. Compared to wild-type C. reinhardtii, recombinant cells overexpressing SAMS grew 1.56-fold faster and produced 1.51-fold more lipids in a nitrogen-depleted medium. Furthermore, under saline-stress conditions, the survival rate and lipid accumulation were 1.56 and 2.04 times higher in the SAMS-overexpressing strain, respectively. These results suggest that the overexpression of SAMS in recombinant C. reinhardtii has high potential in the industrial-scale production of biofuels and various other high-value-added materials.