The xylA gene from a marine bacterium, Vibrio sp. strain XY-214, encoding D-xylose isomerase (XylA) was cloned and expressed in Escherichia coli. The xylA gene consisted of 1,320-bp nucleotides encoding a protein of 439 amino acids with a predicted molecular weight of 49,264. XylA was classified into group II xylose isomerases. The native XylA was estimated to be a homotetramer with a molecular mass of 190 kDa. The purified recombinant XylA exhibited maximal activity at 60°C and pH 7.5. Its apparent K (m) values for D-xylose and D-glucose were 7.93 and 187 mM, respectively. Furthermore, we carried out D-xylulose production from β-1,3-xylan, a major cell wall polysaccharide component of the killer alga Caulerpa taxifolia. The synergistic action of β-1,3-xylanase (TxyA) and β-1,3-xylosidase (XloA) from Vibrio sp. strain XY-214 enabled efficient saccharification of β-1,3-xylan to D-xylose. D-xylose was then converted to D-xylulose by using XylA from the strain XY-214. The conversion rate of D-xylose to D-xylulose by XylA was found to be approximately 40% in the presence of 4 mM sodium tetraborate after 2 h of incubation. These results demonstrated that TxyA, XloA, and XylA from Vibrio sp. strain XY-214 are useful tools for D-xylulose production from β-1,3-xylan. Because D-xylulose can be used as a source for ethanol fermentation by yeast Saccharomyces cerevisiae, the present study will provide a basis for ethanol production from β-1,3-xylan.
The β-1,3-xylosidase gene (xloA) of Vibrio sp. strain XY-214 was cloned and expressed in Escherichia coli. The xloA gene consisted of a 1,608-bp nucleotide sequence encoding a protein of 535 amino acids with a predicted molecular weight of 60,835. The recombinant β-1,3-xylosidase hydrolyzed β-1,3-xylooligosaccharides to d-xylose as a final product.
PhiC31 integrase has the potential to achieve long-term transgene expression because of site-specific and unidirectional recombination. In this study, plasmid DNA (pDNA) encoding Gaussia luciferase (Gluc) cDNA was constructed and the optimal condition for long-term gene expression using phiC31 integrase in mouse liver after hydrodynamic injection was investigated. Gluc is secreted and thus allows quantification of its expression level in blood and easy determination of the expression time-course. Mice were co-transfected with 25 µg donor pDNA (pORF-Gluc/attB) and different amounts of helper pDNA (pCMV-int; from 1 to 50 µg). Serum Gluc expression was evaluated during 120 d. The most highly sustained gene expression was obtained when 10 µg of helper pDNA was co-transfected in ICR and C57BL/6 mice. However, the Gluc expression in C57BL/6 mice was slightly lower and less durable than that in ICR mice. Little hepatic damage caused by phiC31 integrase was observed during 120 d in ICR and C57BL/6 mice.Key words non-viral gene delivery; gene vector; plasmid DNA; hepatocyte A variety of strategies for long-term gene expression have been developed. However, many obstacles that hinder safe and effective gene therapy remain. For example, retroviral vectors, which induce highly efficient integration, mediate random integration into genomic DNA that activated oncogenes both in animals and in clinical trials.1-3) Transposons have been used as an effective method of genomic integration in vivo, but they also integrate randomly.4) Therefore, the use of site-specific recombinases such as Cre and Flp could be a promising method; however, their main utility is in creating deletions mediated by the reversibility of their enzyme reactions. 5,6) It was reported that bacteriophage phiC31 integrase performs a site-specific and unidirectional reaction. 7) In nature, phiC31 integrase mediates the recombination of the phage genome into the bacterial chromosome via a reaction between the phage attP site and the bacterial attB site. 8) Recently, Thyagarajan et al. have demonstrated that phiC31 integrase can act in cultured mammalian cells and that it can efficiently recombine plasmid DNA (pDNA) that includes an attB site into so called pseudo attP sites in mammalian genomic DNA.9) In addition, the number of potential sites integrated by phiC31 integrase in the human genome was estimated at ca. 370 sites, and the sites appear to be in comparatively safe locations.
10)So far, the use of phiC31 integrase for prolonged gene expression has been demonstrated in mice [11][12][13] and in cultured cells. 9,14) This system requires the use of two types of pDNAs: the donor pDNA encoding attB and the helper pDNA encoding phiC31 integrase. Hydrodynamic injection is a transfection method that permits high gene expression in the liver and there are many reports combining this method with phiC31 integrase for basic research using firefly luciferase, genes related to genetic diseases or hepatic murine cancer model. 11,[15][16][17][18][19][20][21] Taking thes...
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