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 digested with NcoI and SacI and cloned into the respective sites of pET52b(+), giving rise to pET52bMamAΔ41-AMB1. In this construct, the mamAΔ41 gen....

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 .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
French Press	Equipment	Thermo Fisher Scientific, Inc.	FA-078A
Pressure cell	Equipment	Thermo Fisher Scientific, Inc.	FA-032
Ultra-centrifuge	Equipment	Sorvall, Thermo Scientific	Discovery 90SE
Rottor	Equipment	Beckman Coulter Inc.	Ti60
Ultra-centrifuge tubes; PC-Bottle+Cap Assay 26.3ml	Equipment	Beckman Coulter Inc.	BC-355618
2.5cm diameter, Glass Econo-Column Chromatography Columns	Equipment	Bio-Rad	737-2521
Ni-NTA His Bind resin	Equipment	Novagen, EMD Millipore	M0063428
Spectrophotometer	Equipment	Amersham	Ultraspec 2100 pro
Quartz cuvette	Equipment	Hellma	104-QS
Fast Performance Liquid Chromatography- AKTA purifier 10	Equipment	GE Healthcare	28-4062-64
Ion exchange column – MonoQ 4.6/100 PE	Equipment	GE Healthcare	10025543
Size exclusion pre-packed column-HiLoad 26/60 Superdex 200	Equipment	GE Healthcare	17-1071-01
Centricon - Vivaspin15 – 10,000 MWCO	Equipment	Sartorius AG	VS1501
Table centrifuge	Equipment	Thermo Fisher Scientific, Inc.	IEC CL30R
MALDI-TOF	Equipment	Bruker Corporation	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-Aldrich	P-8849
Dnase I (Deoxyribonuclease I)	Reagent	Sigma-Aldrich	DN-25
Bovine Thrombin	Reagent	Fisher Scientific	BP25432
Glycine	Reagent	BioLab	07132391
Soudim Dodecyl Sulfate (SDS)	Reagent	BioLab	19822391
Beta-mercapt–thanol	Reagent	Sigma-Aldrich	M-3148
InstantBlue	Reagent	Expedeon	1SB01L
PageRuler Prestained Protein Ladder	Reagent	Fermentas	SM0671