| Budget Amount *help |
¥6,900,000 (Direct Cost: ¥6,900,000)
Fiscal Year 1994: ¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1993: ¥4,900,000 (Direct Cost: ¥4,900,000)
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| Research Abstract |
Magnetospirillum sp. AMB-1 is a freshwater magnetic bacterium which synthesizes intracellular particles of magnetite (Fe_30_4). Five nonmagnetic mutants of AMB-1 were previously generated by introduction of transposon Tn5.
In this study, a genomic DNA fragment required for synthesis of magnetic particles was isolated from one of the nonmagnetic mutants, NM5. We have determined the complete nucleotide sequence of this fragment. The 2975 bp region contains two putative open reading frames. One ORF,designated magA,encodes a polypeptide which is homologous to the cation efflux proteins, the Escherichia coli potassium ion translocating protein, KefC,and the putative Na^+/H^+-antiporter, NapA,from Enterococcus hirae. Northern hybridization demonstrated that the magA mRNA transcript is 1.3 kb in size, corresponding to the size of the magA gene. A functional promoter was located upstream from the magA gene, and the transcription was regulated by environmental iron concentration in AMB-1.
We devel ped a simple and general method in which peptides can be displayd on the surface of bacterial magnetic particles facilitating their direct magnetic purification using the magA gene fusion. Genes encoding the proteins to be displayd are fused downstream from the Magnetospirillum magA gene which are then expressed on the magnetic particle surface. To demonstrate the effectiveness of this method firefly luciferase was expressed on the surface of the magnetic particles which could then be recovered by cell lysis and magnetic purification. Luciferase activity was detected on the magnetic particles which were isolated from AMB-1 harboring the magA-luciferase fusion gene. This approach will significantly enhance protein screening and purification strategies.
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