Production of luciferase-magnetic particle complex by recombinant Magnetospirillum sp. AMB-1.
Luciferase-bacterial magnetic particle (BMP) complexes were produced by recombinant Magnetospirillum sp. AMB-1. We constructed plasmids pKML and pNELM, respectively, by fusing luc to the 5' and 3' terminal of magA, encoding an integral iron translocating protein situated in the BMP membrane, of AMB-1. In addition, we produced bifunctional active-fusion proteins on BMPs by using a plasmid pAcML. In this plasmid, acetate kinase and luciferase genes were fused to the N-terminus and the C-terminus of MagA, respectively. Bacterial magnetic particles isolated from transconjugants for pKML, pNELM and pAcML exhibited luciferase activity. Bacterial magnetic particles isolated from transconjugants for pAcML also exhibited acetate kinase activity. Fed-batch culture of pKML transconjugant yielded 2.6 mg BMPs per liter of culture, and 95% conversion of iron into magnetite was obtained, at a nitrate concentration of 1.4 mM. Continuous feeding of iron as ferric quinate significantly enhanced growth and total magnetic production. Final cell concentration of 1.8 x 10(9) cells/mL and 6 mg per liter of culture was obtained. Magnetite production by fed-batch culture of AMB-1 was about 3 times that obtained by batch culture. There were no significant differences in BMPs yield between recombinant AMB-1 cultivated by fed-batch culture and wild type of AMB-1.
Matsunaga
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Department of Biotechnology, Tokyo University of Agriculture and Technology, 2-24-16, Naka-cho, Koganei, Tokyo 184-8588, Japan. tmatsuna@cc.taut.ac.jp
Togo
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Kikuchi
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Tanaka
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D000084:Acetate Kinase / Q000378:metabolism; D001426:Bacterial Proteins; D001709:Biotechnology / Q000379:methods; D002352:Carrier Proteins / Q000378:metabolism; D027682:Cation Transport Proteins; D002455:Cell Division; D002851:Chromatography, High Pressure Liquid; D007118:Immunoassay; D008156:Luciferases / Q000096:biosynthesis*; D008280:Magnetics; D008565:Membrane Proteins / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012244:Rhodospirillaceae / Q000201:enzymology*; D013997:Time Factors
D001426:Bacterial Proteins; D002352:Carrier Proteins; D027682:Cation Transport Proteins; C097085:MagA protein, Magnetospirillum; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D008156:Luciferases; D000084:Acetate Kinase