Purification of the M. magneticum Strain AMB-1 Magnetosome Associated Protein MamAΔ41
Summary
MamA is a unique Magnetosome associated protein which was shown to be involved in magnetosome activation. Here we present the purification protocol of MamA deletion mutant (MamAΔ41) from M. magneticum AMB-1.
Abstract
Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is believed to aid their search for suitable environments (1). This capability is conferred by the magnetosome, a subcellular organelle that consists of a linear-chain assembly of lipid vesicles each able to biomineralize and enclose a ~50-nm crystal of magnetite or greigite. A principle component of the magnetosome that was shown to be required for the formation of functional vesicles is MamA. MamA is a highly abundant magnetosome-associated protein which is one of the most characterized magnetosome-associated proteins in vivo (2-6). This article focuses on the purification of MamA, which despite being studied in vivo, no clear functional or structural details have been identified for it. Bioinformatics analysis suggested that MamA is a tetra-tricopeptide repeat (TPR) containing protein. TPR is a structural motif found as such or forming part of a bigger fold in a wide range of proteins, it serves as a template for protein-protein interactions and mediates multi-protein complexes (7). TPRs are involved in many crucial tasks in eukaryotic cell organelle processes and many bacterial pathways (8-14). In order to understand MamA, a unique TPR containing protein, highly purified protein is required as a first step. In this article, we present the purification protocol for a stable MamA deletion mutant (MamAΔ41) from M. magneticum AMB-1.
Protocol
1. Cloning and Expression of mamA Gene in E. coli The mutant gene mamAΔ41 was amplified using the polymerase chain reaction (PCR) from genomic DNA of Magnetospirillum magneticum AMB-1, with primers: 5′-GCATTACGCATATGGACGACATCCGCCAGGTG-3′ and 5′-GCGCGGCAGCCATA-TGGCATACG-3′. In the amplified DNA fragments, an NcoI site was introduced at the initiation codon ATG and the termination codon was replaced with a ScoI site. The fragments were d…
Discussion
Protein purification is the main step in any proteins biochemical or structural studies. Since each protein is unique with its own behavior, one needs to define its properties and modify its purification accordingly. Protein target should be analyzed as a first step toward purification using bioinformatics tools. They are used to calculate the target iso-electric point, assess its need for reducing/oxidizing environment and its need for special ions/ligands. There are several critical modifications of our protocol which …
Acknowledgements
We acknowledge Dr. Amir Aharoni for his support and Geula Davidov, Noam Grimberg and Chen Guttman for their advice and comments.
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 |
Riferimenti
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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).