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Purification and initial characterization of the H+/Mg2+ ATPase B from Salmonella typhimurium

Toushik, Md Sazzad Hossen
Master thesis
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Purification and initial characterization of the H+-Mg2+ ATPase B from s. typhimurium-Toushik.pdf (33.28Mb)
URI
http://hdl.handle.net/11250/186610
Date
2014-02-18
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  • Master's theses (KBM) [742]
Abstract
The structural determination of MgtB is not only necessary to reveal its unknown mechanism but also would be of great value to compare with other Mg2+ transporters in the Mg2+ transport system. Like most gram negative bacteria, Salmonella typhimurium also possesses MgtB transporter in Mg2+ transport system. The gene mgtB encodes the ~100.0 kDa MgtB protein that is considered a P-type ATPase. The protein sequence shows more similarity with the eukaryotic P-type ATPase family members compares to the family members of prokaryotic P-type ATPase. It is assumed that, MgtB transports Mg2+ across the plasma membrane to counteract reduced Mg2+ concentrations under growth conditions along with other transporters in Mg2+ transport system. The experiments that are described in this thesis were performed to characterize the Mg2+ transporter protein, MgtB from S. typhimurium by using expression, purification and crystallization techniques.

Purification was optimized of the recombinant protein MgtB; the experimental result gives an idea about the buffer, pH and salt concentrations in which MgtB was more stable. The result showed that MgtB is more stable in 10 mM Tris-HCl pH 7.6 and 100 mM NaCl concentration. It was also observed that the lipid bilayers of MgtB protein was adequately solubilized by the detergent DDM, however during purification the detergent C12E8 showed better analytical result. No protein crystals were observed from MgtB crystal setup in this study, even though some crystal was observed in some conditions but it turned out to be either salt or detergent. We assumed that this could have happened because of protein aggregation and instability. To avoid this problem in the future, additional experiments that include lipidation and biochemical characterization has been suggested to achieve a crystal structure of MgtB.
Publisher
Norwegian University of Life Sciences, Ås

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