Comparative study and expression analysis of the interferon gamma gene locus cytokines in Xenopus tropicalis

Using bioinformatics approach, the genome locus containing interleukin (IL)-22, IL-26, and interferon gamma (IFN-gamma) genes has been identified in the amphibian, Xenopus tropicalis. Like that in other vertebrates such as fish, birds, and mammals, the Xenopus IL-22, IL-26, and IFN-gamma are clustered in the same chromosome and the adjacent genes are conserved. The genomic structures of the Xenopus IL-22, IL-26, and IFN-gamma gene were identical to that of their mammalian counterparts. The Xenopus IL-22 and IL-26 genes contained five exons and four introns while the Xenopus IFN-gamma gene consisted of four exons and three introns. The Xenopus IL-22, IL-26, and IFN-gamma share 14.1-41.6%, 14.6-31.2%, and 23.7-36.5% identity to their counterparts in other species, respectively. Reverse-transcription polymerase chain reaction (PCR) and real-time quantitative PCR analyses revealed that the expression of IL-22, IL-26, and IFN-gamma genes was significantly upregulated after simulation with bacterial polyliposaccharide and/or synthetic double-stranded poly(I:C), suggesting these cytokines like those in other vertebrates play an important role in regulating immune response in Xenopus.

Biosynthesis of gold nanoparticles assisted by Escherichia coli DH5 alpha and its application on direct electrochemistry of hemoglobin

In this communication, biosynthesis of gold nanoparticles assisted by Escherichia coli DH5 alpha and its application on direct electrochemistry of hemoglobin are reported. The gold nanoparticles formed on the bacteria surface are mostly spherical. The direct electrochemistry of hemoglobin can be achieved by incorporated into the bio-nanocomposite films on a glassy carbon electrode.

Functional biosynthesis of an allophycocyan beta subunit in Escherichia coli

Allophycocyanin is a phycobiliprotein with various biological and pharmacological properties. An expression vector was constructed using CpeS as the bilin lyase for the allophycocyanin beta subunit, resulting in overexpression of a fluorescent allophycocyanin beta-subunit in Escherichia coli. A high-density cell culture was developed using a continuous feeding strategy. After 16 h of culture, the dry cell density reached 21.4 g 1(-1), the expression of the allophycocyanin beta-subunit was 0.86 g l(-1) broth, and the relative chromoprotein yield was 81.4%. The recombinant protein showed spectral features similar to native allophycocyanin, which provide an efficient methodology for large-scale production of this valuable fluorescent protein. (C) 2008, The Society for Biotechnology, Japan. All rights reserved.

Biosynthesis of fluorescent allophycocyanin alpha-subunits by autocatalysis in Escherichia coli

APC (allophycocyanin) is widely used for fluorescence tagging and may be a promising antioxidant agent for use within the food and pharmaceutical industries. Chromophore attachment to apo-ApcA (apo-APC alpha-subunit without chromophore) can be auto-catalysed both in vitro and in vivo. In the present study, a plasmid containing genes of apo-ApcA and chromophore synthetases (HOI (ferredoxin-dependent haem oxygenase) and PcyA (phycocyanobilin:ferredoxin oxidoreductase)] was constructed and expressed in Escherichia coli. The results show that holo-ApcA (APC alpha-subunit with chromophore) can be synthesized by autocatalysis in E. coli. Recombinant holo-ApcA showed the same spectral and fluorescent properties as PC (phycocyanin) and could serve as a good substitute for native PC for fluorescent tagging. Moreover, recombinant ApcA can inhibit hydroxyl and peroxyl radicals more strongly than holo-ApcA and native APC. The EC50 values were 296.4 +/- 22.4 mu g/ml against hydroxyl radicals and 38.5 +/- 2.6 mu g/ml against peroxyl radicals.

Positional distribution of fatty acids on the glycerol backbone during the biosynthesis of glycerolipids in Ectocarpus fasciculatus

The biosynthesis of glycolipids in E. fasciculatus was studied by C-14 label and chase. The fatty acids in sulphoquinovosyl diacylglycerol (SQDG) were almost 16-carbon and 18-carbon ones. In addition to the two fatty acids, monogalactosyl diacylglycerol (MGDG) and digalactosyl diacylglycerol (DGDG) contained 8.5 mol% and 31.0 mol% of eicosapentaenoic acid (20 : 5), respectively, and this fatty acid was usually distributed in the sn-1 position of the glycerol backbone. When plants were incubated with [2-C-14] acetate, differences existed in the positional distribution of the labeled fatty acids in sn-1 and sn-2 among the three glycerolipids. In SQDG C-14-labeled fatty acids were distributed uniformly in the sn-1 and sn-2 positions. In DGDG, C-14-labeled fatty acids were mainly distributed in the sn-2 position. In MGDG, the radioactivity of fatty acids in sn-1 position was far greater than that in sn-2 position after a 30 min pulse label, and the difference in radioactivity between the two positions decreased rapidly. The above results indicated that differences in the positional distribution of C-14-labeled fatty acids between sn-1 and sn-2 positions might be related to 20 : 5 and the biosynthesis of DGDG. Our results also suggested that E. fasciculatus had the same DGDG biosynthetic pathway as that in higher plants and galactosyl transferase was selective for MGDC.

Fatty acid biosynthesis in eukaryotic photosynthetic microalgae: Identification of a microsomal delta 12 desaturase in Chlamydomonas reinhardtii

Polyunsaturated fatty acids (PUFAs) are important components of infant and adult nutrition because they serve as structural elements of cell membranes. Fatty acid desaturases are responsible for the insertion of double bonds into pre-formed fatty acid chains in reactions that require oxygen and reducing equivalents. In this study, the genome-wide characterization of the fatty acid desaturases from seven eukaryotic photosynthetic microalgae was undertaken according to the conserved histidine-rich motifs and phylogenetic profiles. Analysis of these genomes provided insight into the origin and evolution of the pathway of fatty acid biosynthesis in eukaryotic plants. In addition, the candidate enzyme from Chlamydomonas reinhardtii with the highest similarity to the microsomal Delta 12 desaturase of Chlorella vulgaris was isolated, and its function was verified by heterologous expression in yeast (Saccharomyces cerevisiae).

Combinational biosynthesis of a fluorescent cyanobacterial holo-alpha-allophycocyanin in Escherichia coli

Allophycocyanin ( APC) is a phycobiliprotein with various biological and pharmacological properties. An expression vector containing five essential genes in charge of biosynthesis of cyanobacterial APC holo-alpha subunit ( holo- ApcA) was constructed, resulting in over- expression of a fluorescent holo- ApcA in E. coli. After being cultured for 16 h, the dry cell density reached 22.5 gl(-1), and the expression of holo- HT- ApcA was up to 1 gl(-1) broth. The recombinant protein showed similar spectral features to native APC.

Biosynthesis of fluorescent cyanobacterial allophycocyanin trimer in Escherichia coli

Allophycocyanin (APC), a cyanobacterial photosynthetic phycobiliprotein, functions in energy transfer as a light-harvesting protein. One of the prominent spectroscopic characteristics of APC is a strong red-shift in the absorption and emission maxima when monomers are assembled into a trimer. Previously, holo-APC alpha and beta subunits (holo-ApcA and ApcB) were successfully synthesized in Escherichia coli. In this study, both holo-subunits from Synechocystis sp. PCC 6803 were co-expressed in E. coli, and found to self-assemble into trimers. The recombinant APC trimer was purified by metal affinity and size-exclusion chromatography, and had a native structure identical to native APC, as determined by characteristic spectroscopic measurements, fluorescence quantum yield, tryptic digestion analysis, and molecular weight measurements. Combined with results from a study in which only the monomer was formed, our results indicate that bilin synthesis and the subsequent attachment to apo-subunits are important for the successful assembly of APC trimers. This is the first study to report on the assembly of recombinant ApcA and ApcB into a trimer with native structure. Our study provides a promising method for producing better fluorescent tags, as well as a method to facilitate the genetic analysis of APC trimer assembly and biological function.