ISSN: 1449-2288International Journal of Biological Sciences
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Int J Biol Sci 2008; 4(2):71-80. doi:10.7150/ijbs.4.71 This issue Cite
Research Paper
Shedding Light on the Role of Vitreoscilla Hemoglobin on Cellular Catabolic Regulation by Proteomic Analysis
1. Department of Clinical Microbiology, Faculty of Medical Technology, Mahidol University, Bangkok 10700, Thailand,
2. Department of Medical Technology, Faculty of Allied Health Science, Thammasat University, Pathum Thani 12120, Thailand
Abstract
Heterologous expression of Vitreoscilla hemoglobin (VHb) has been reported to improve cell growth, protein synthesis, metabolite productivity and nitric oxide detoxification. Although it has been proposed that such phenomenon is attributed to the enhancement of respiration and energy metabolism by facilitating oxygen delivery, the mechanism of VHb action remains to be elucidated. In the present study, changes of protein expression profile in Escherichia coli as a consequence of VHb production was investigated by two-dimensional gel electrophoresis (2-DE) in conjunction with peptide mass fingerprinting. Total protein extracts derived from cells expressing native green fluorescent protein (GFPuv) and chimeric VHbGFPuv grown in Luria-Bertani broth were prepared by sonic disintegration. One hundred microgram of proteins was individually electrophoresed in IEF-agarose rod gels followed by gradient SDS-PAGE gels. Protein spots were excised from the gels, digested to peptide fragments by trypsin, and analyzed using matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry. Results revealed that expression of VHbGFPuv caused an entire disappearance of tryptophanase as well as down-regulated proteins involved in various metabolic pathways, e.g. glycerol kinase, isocitrate dehydrogenase, aldehyde dehydrogenase, and D-glucose-D-galactose binding protein. Phenotypic assay of cellular indole production confirmed the differentially expressed tryptophanase enzymes in which cells expressing chimeric VHbGFP demonstrated a complete indole-negative reaction. Supplementation of δ-aminolevulinic acid (ALA) to the culture medium enhanced expression of glyceraldehyde-3-phosphate dehydrogenase and glycerol kinase. Our findings herein shed light on the functional roles of VHb on cellular carbon and nitrogen consumptions as well as regulation of other metabolic pathway intermediates, possibly by autoregulation of the catabolite repressor regulons.
Keywords: Vitreoscilla hemoglobin (VHb), Two-dimensional gel electrophoresis (2-DE), Proteomic, Catabolic regulation, Peptide Mass Fingerprinting (PMF), Mass spectrometry
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