权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Modify of the bacterial magnetic particles by gene manipulation

通过基因操作修饰细菌磁性颗粒

基本信息

批准号：
05453109
负责人：
MATSUNAGA Tadashi
金额：
$ 4.42万
依托单位：
Tokyo University of Agriculture and Technology
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1994
项目状态：
已结题

项目摘要

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.

磁细菌AMB-1是一种淡水磁性细菌，能合成磁铁矿（Fe_3O_4）。通过转座子Tn 5的导入，获得了5个AMB-1的转座子突变体，本研究从其中一个转座子突变体NM 5中分离到了合成磁性颗粒所需的基因组DNA片段。我们已经确定了该片段的完整核苷酸序列。2975 bp的区域包含两个推定的开放阅读框架。其中一个开放阅读框命名为magA，它编码的多肽与阳离子外排蛋白、大肠杆菌钾离子转运蛋白KefC和肠球菌Na^+/H^+反向转运蛋白NapA同源。北方杂交表明，magA mRNA转录本的大小为1.3 kb，对应于magA基因的大小。在AMB-1中，magA基因上游有一个功能性启动子，其转录受环境铁浓度的调控。我们开发了一种简单而通用的方法，利用magA基因融合技术，将多肽展示在细菌磁性颗粒表面，从而直接进行磁性纯化。编码待展示的蛋白质的基因融合在磁细菌magA基因的下游，然后在磁性颗粒表面上表达。为了证明该方法的有效性，在磁性颗粒的表面上表达萤火虫荧光素酶，然后可以通过细胞裂解和磁性纯化来回收所述磁性颗粒。荧光素酶活性在从携带magA-荧光素酶融合基因的AMB-1分离的磁性颗粒上检测。这种方法将显著增强蛋白质筛选和纯化策略。