Published: Vol 6, Iss 22, Nov 20, 2016 DOI: 10.21769/BioProtoc.2001 Views: 10206
Reviewed by: Arsalan DaudiKanika GeraChijioke Joshua
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Abstract
The magnesium transporter A (MgtA) is a magnesium transporting P-type ATPase present in prokaryotes and plants (Subramani et al., 2016). In Salmonella typhimurium and Escherichia coli (E. coli), MgtA is expressed only in magnesium limiting conditions and plays an important role in Mg2+ homeostasis (Groisman et al., 2013). The transcription of mgtA is regulated by the two-component system PhoP/PhoQ (Soncini et al., 1996; Kato et al., 1999). The membrane bound histidine kinase, PhoQ, senses low Mg2+ concentration in the periplasmic space and phosphorylates its cognate response regulator, PhoP, which initiates mgtA transcription (Groisman et al., 2013). MgtA is targeted to the plasma membrane and facilitate the bacterial survival under low Mg2+ condition, by importing Mg2+ into the cytoplasm. The MgtA homolog in petunia (PH1) is found in the vacuolar membrane and involved with the coloration of the flower petals (Faraco et al., 2014). As a first step towards understanding the molecular details of MgtA Mg2+ transport, we describe a detailed protocol for the purification of E. coli MgtA that can be used for biochemical and biophysical studies. Recombinant E. coli MgtA with hexa histidine tag at the N-terminus was cloned from E. coli DH5α and over expressed in the E. coli C43(DE3) by fermentation to an OD > 6. Cell lysis was performed in a high pressure homogenizer and the membranes were isolated by ultracentrifugation. Membrane proteins were solubilized with the detergent dodecyl-β-D maltoside. MgtA was purified by affinity and size exclusion chromatography. Final yields of purified MgtA reach ~1 mg MgtA per 3 g of wet cell pellet.
Background
Recently we have reported that the purified MgtA from E. coli is highly dependent on lipids for its function and studied the enzyme kinetics in vitro (Subramani et al., 2016). The protocol described here is the detailed description going through every single step of the purification that should yield monodisperse detergent solubilized MgtA, earlier described in Subramani et al. (2016). The protocol will in addition present notes that describe critical points and observations made in the process.
Materials and Reagents
Equipment
Software
Procedure
Data analysis
The data for chromatograms in Figure 1 were obtained from UNICORN software associated with ÄKTA purifier and the graphs were plotted using GraphPad Prism6. All the SDS-PAGE gels were scanned using Bio-Rad ChemiDoc XRS+ system and analyzed using Image LabTM software.
Recipes
Note: The buffers used for chromatography were always filtered using the NalgeneTM reusable bottle top filter connected to vacuum pump. Buffers were degassed with the same set up, but by closing the filter chamber while the vacuum still on and with constant stirring. Buffers were degassed until no bubbles were observed in the solution.
Acknowledgments
The Norwegian Research Council Funding (F-RIMEDBIO) #ES486454 and NCMM core Funding supported this study.
References
Article Information
Copyright
Subramani and Morth. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
How to cite
Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
Category
Microbiology > Microbial biochemistry > Protein
Biochemistry > Protein > Expression
Biochemistry > Protein > Isolation and purification
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