Authors: Shelake Rahul Mahadev Hidenori Hayashi Takahisa Ikegami Shunnosuke Abe Eugene Hayato Morita
Publish Date: 2013/11/22
Volume: 32, Issue: 8, Pages: 626-634
Abstract
There are structural and functional differences in SmtB homologs metalresponsive transcription factors responsible for sensing of excess heavy metal ions in marine and freshwater cyanobacterial strains The structure of SmtB from freshwater Synechococcus sp strain PCC 7942 is elucidated with nuclear magnetic resonance NMR and crystallography techniques But knowledge about the functioning of SmtB homologs from marine species is limited till date To enable NMR spectroscopic studies for investigating structural and functional aspects modified protocols with higher yields of isotopically labeled SmtB from marine species like Synechococcus sp PCC 7002 are essential In this study smtB gene was cloned from genome of Synechococcus sp PCC 7002 and overexpression protocol for recombinant SmtB was standardized in Escherichia coli containing T7 RNA polymerase/promoter system Further the protocol for largescale production isotope labeling with 15N and purification of recombinant SmtB in E coli BL21DE3/pLysS cells was developed Purified recombinant protein was successfully used for NMR spectroscopy experiments These results indicate that the overexpression technique now developed is applicable to the structural and functional studies for the proteins being homologous to cyanobacterial SmtB from Synechococcus sp PCC 7002This research was partially supported by MEXT Ministry of Education Culture Sports Science and Technology in Japan a GrantinAid for scientific research C 22510234 2010–2012 to EHM and Doctoral fellowship to RMS Authors are grateful to the staff of Integrated Center for Sciences Tarumi Ehime University for providing nucleotide sequencing facility
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