• BMB Reports
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• v.33 no.3
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• pp.195-201
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• 2000

A human gene has been reported that may encode the enzyme acetohydroxyacid synthase. Previously this enzyme was thought to be absent from animals although it is present in plants and many microorganisms. In plants, this enzyme is the target of a number of commercial herbicides and the use of these compounds may need to be reassessed if the human enzyme exists and proves to be susceptible to inhibition. Here we report the construction of several plasmid vectors containing the cDNA sequence for this protein, and their expression in Escherichia coli. High levels of expression were observed, but most of the protein proved to be insoluble. The small amounts of soluble protein contained little or no acetohydroxyacid synthase activity. Attempts to refold the insoluble protein were successful insofar as the protein became soluble. However, the refolded protein did not gain any acetohydroxyacid synthase activity. In vivo complementation tests of an E. coli mutant produced no evidence that the protein is active. Incorrect folding, or the lack of another subunit, may explain the data but we favor the interpretation that this gene does not encode an acetohydroxyacid synthase.

• BMB Reports
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• v.28 no.1
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• pp.57-61
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• 1995

The expression of a recombinant human lactoferrin is reported in mouse HC11 mammary epithelial cells. Expression of human lactoferrin (hLF) was achieved by placing its cDNA under the control of the bovine ${\beta}$-casein gene. To improve the hLF expression level in a cell culture system, two artificial introns were also introduced to construct expression vectors. One intron was a hybrid-splice signal consisting of bovine ${\beta}$-casein intron 1 and rabbit ${\beta}$-globin intron II. The other intron was a DNA fragment spanning intron 8 of the bovine ${\beta}$-casein gene. The hybrid intron moderately elevated hLF expression, whereas intron 8 alone did not express any detectable amount of hLF as judged by Northem and Western blot analyses. When the two introns were used together they contributed to a synergistic elevation of hLF expression. These data indicate that artificial introns on both sides of the hLF cDNA were necessary to increase expression of cDNA.

• Toxicological Research
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• v.18 no.3
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• pp.249-257
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• 2002

Cytochrome P450 3As such as 3A4 and 3A5 metabolize a wide range of pharmaceutical compounds. The vectors for the expression of fusion protein containing an N-terminal human P450 3A4 or P450 3A5 sequences and a C-terminal rat NADPH-cytochrome P450 reductase moiety were constructed. These plasmids were used to express the fusion protein in Escherichia coli DH5$\alpha$ cells. High levels of expression were achieved (100~200 nmol/liter) and the expressed fusion protein in E. coli membranes were catalytically active for nifedipine oxidation, a typical enzymatic activity of P450 3A4. The NADPH-P450 reductase activities of these fusion protein were also determined by measuring reduction of cytochrome c. To fine a specific Inhibitor of P450 3A4 from naturally occurring chemicals, a series of isothiocyanate compounds were evaluated for the inhibitory activity of P450 using the fusion proteins in E. coli membranes. Of the five isothiocyanates (phenethyl isothiocyanate, phenyl isothiocyanate, benzol isothiocyanate, benzoyl isothiocyanate and cyclohexyl isothiocyanate) tested, benzoyl isothiocyanate showed a strong inhibition of P450 3A4 with an $IC_{50}$value of 2.8 $\mu\textrm{M}$. Our results indicate that the self-sufficient fusion protein will be very useful tool to study the drug metabolism and benzyl isothiocyanate may be valuable for characterizing the enzymatic properties of P450 3A4.

• KSBB Journal
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• v.13 no.2
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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.

• Journal of Microbiology and Biotechnology
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• v.32 no.3
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• pp.278-286
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• 2022

Live bacterial vector vaccines are one of the most promising vaccine types and have the advantages of low cost, flexibility, and good safety. Meanwhile, protein secretion systems have been reported as useful tools to facilitate the release of heterologous antigen proteins from bacterial vectors. The twin-arginine translocation (Tat) system is an important protein export system that transports fully folded proteins in a signal peptide-dependent manner. In this study, we constructed a live vector vaccine using an engineered commensal Escherichia coli strain in which amiA and amiC genes were deleted, resulting in a leaky outer membrane that allows the release of periplasmic proteins to the extracellular environment. The protective antigen proteins SLY, enolase, and Sbp against Streptococcus suis were targeted to the Tat pathway by fusing a Tat signal peptide. Our results showed that by exploiting the Tat pathway and the outer membrane-defective E. coli strain, the antigen proteins were successfully secreted. The strains secreting the antigen proteins were used to vaccinate mice. After S. suis challenge, the vaccinated group showed significantly higher survival and milder clinical symptoms compared with the vector group. Further analysis showed that the mice in the vaccinated group had lower burdens of bacteria load and slighter pathological changes. Our study reports a novel live bacterial vector vaccine that uses the Tat system and provides a new alternative for developing S. suis vaccine.