纯化 M。 magneticum应变AMB - 1磁小体相关蛋白MamAΔ41
Summary
妈妈是一个独特的磁相关蛋白被证明是参与磁激活。在这里，我们从目前的妈妈缺失突变体的净化协议（MamAΔ41）<em> M。 magneticum</em> AMB – 1。
Abstract
磁细菌组成的水生微生物，能够沿着地磁场的方向不同群体。这种行为被认为是帮助他们寻找合适的环境<em>（1）</em>。这种能力是由磁赋予的，一个细胞内的细胞器，包含一个线性链大会脂质囊泡每个能够biomineralize，并附上了〜50纳米磁铁矿或胶黄铁矿晶体。一个原则的功能囊泡的形成，被证明是需要的磁组件是妈妈。妈妈是一个非常丰富的磁相关的蛋白质，这是最特征的磁相关蛋白之一<em>在体内</em><em>（2-6）</em>。本文的重点，在妈妈的净化，尽管正在研究<em>在体内</em>，没有明确的功能或结构细节已经确定了它。生物信息学分析表明，妈妈是一个四tricopeptide重复（TPR），含蛋白质。 TPR是在广泛的蛋白质折叠的一部分，或形成一个结构基序，它可作为蛋白质相互作用，介导的多蛋白质复合物的模板<em>（7）</em>。 TPRS参与许多重要的任务，在真核细胞的细胞器的进程和许多细菌途径<em>（8-14）。</em>为了理解妈妈，一个独特的TPR，含有蛋白质，高度纯化的蛋白质是必需的第一步。在这篇文章中，我们目前为一个稳定的妈妈缺失突变体的净化（MamAΔ41）协议<em> M。 magneticum</em> AMB – 1。
Protocol
1。 妈妈在大肠杆菌中的基因的克隆和表达大肠杆菌 突变基因mamAΔ41Magnetospirillum magneticum AMB – 1的基因组DNA扩增的聚合酶链反应（PCR）引物：5' – GCATTACGCATATGGACGACATCCGCCAGGTG 3'和5' – GCGCGGCAGCCATA – TGGCATACG – 3“ 。在扩增的DNA片段，NcoI网站介绍，在起始密码子ATG和终止密码子与ScoI网站所取代。片段，NcoI和SACI消化和克隆到pET52b（+），在各自的…
Discussion
蛋白质纯化的生化或在任何蛋白质结构研究的主要步骤。由于每个蛋白质独特的是与自己的行为，需要定义其属性，并相应修改其净化。分析蛋白质的目标应该是，作为对使用净化生物信息学工具的第一步。它们被用来计算目标ISO电点，评估其需要减少/氧化环境，它需要特殊的离子/配体。我们的协议中有几个关键的修改反映了目标的独特性。这些修改包括缓冲区，工作温度和列调整。
<p class=…Acknowledgements
我们承认对他的支持和对他们的建议和意见，诺姆Grimberg Geula达维多夫和陈格特曼博士阿米尔Aharoni。
Materials
| Material Name | Tipo | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| French Press | Equipment | Thermo scientific | FA-078A | |
| Pressure cell | Equipment | Thermo scientific | FA-032 | |
| Ultra-centrifuge | Equipment | Sorvall | Discovery 90SE | |
| Rottor | Equipment | Beckman | Ti60 | |
| Ultra-centrifuge tubes; PC-Bottle+Cap Assay 26.3ml | Equipment | Beckman | BC-355618 | |
| 2.5cm diameter, Glass Econo-Column Chromatography Columns | Equipment | BioRad | 737-2521 | |
| Ni-NTA His Bind resin | Equipment | Novagen | M0063428 | |
| Spectrophotometer | Equipment | Amersham Biosiences | Ultraspec 2100 pro | |
| Quartz cuvette | Equipment | Hellma | 104-QS | |
| Fast Performance Liquid Chromatography- AKTA purifier 10 | Equipment | GE Healthcare Biosciences | 28-4062-64 | |
| Ion exchange column – MonoQ 4.6/100 PE | Equipment | GE Healthcare Biosciences | 10025543 | |
| Size exclusion pre-packed column-HiLoad 26/60 Superdex 200 | Equipment | GE Healthcare Biosciences | 17-1071-01 | |
| Centricon – Vivaspin15 – 10,000 MWCO | Equipment | Sartorius Stedim Biotech GmbH | VS1501 | |
| Table centrifuge | Equipment | Thermo scientific | IEC CL30R | |
| MALDI-TOF | Equipment | Bruker Daltonics | Reflex IV | |
| Tris-HCl (hydrotymethyl) aminomethane | Reagent | BioLab | 20092391 | |
| Sodium Chloride | Reagent | FRUTROM | 235553470 | |
| Imidazole | Reagent | Alfa Aesar | 288-32-4 | |
| EDTA free protease inhibitors cocktail | Reagent | Sigma | P-8849 | |
| Dnase I (Deoxyribonuclease I) | Reagent | Sigma | DN-25 | |
| Bovine Thrombin | Reagent | Fisher BioReagents | BP25432 | |
| Glycine | Reagent | BioLab | 07132391 | |
| Soudim Dodecyl Sulfate (SDS) | Reagent | BioLab | 19822391 | |
| Beta-mercaptoethanol | Reagent | Sigma | M-3148 | |
| InstantBlue | Reagent | Expedeon | 1SB01L | |
| PageRuler Prestained Protein Ladder | Reagent | Fermentas | SM0671 |
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Tags
PurificationM. MagneticumStrain AMB-1Magnetosome Associated ProteinMamAΔ41Magnetotactic BacteriaGeomagnetic FieldsMagnetosomeLipid VesiclesBiomineralizeMagnetiteGreigiteFunctional VesiclesMagnetosome-associated ProteinPurification Of MamAFunctional DetailsStructural DetailsBioinformatics AnalysisTetra-tricopeptide Repeat (TPR)Protein-protein InteractionsMulti-protein ComplexesEukaryotic Cell Organelle ProcessesBacterial Pathways
Citazione di questo articolo
Zeytuni, N., Zarivach, R. Purification of the M. magneticum Strain AMB-1 Magnetosome Associated Protein MamAΔ41. J. Vis. Exp. (37), e1844, doi:10.3791/1844 (2010).