title: Improved protein overexpression and purification strategies for structural studies of cyanobacterial metal-responsive transcription factor, SmtB from marine Synechococcus sp. PCC 7002.
abstract: There are structural and functional differences in SmtB homologs, metal-responsive 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 large-scale production, isotope labeling with (15)N, and purification of recombinant SmtB in E. coli BL21(DE3)/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 7002.
authors.affiliation: Laboratory of Molecular Cell Physiology, Department of Bioresources, Faculty of Agriculture, Ehime University, Matsuyama, 790-8566, Japan.
authors.forename: Shelake Rahul
authors.initials: SR
authors.lastname: Mahadev
authors.forename: Hidenori
authors.initials: H
authors.lastname: Hayashi
authors.forename: Takahisa
authors.initials: T
authors.lastname: Ikegami
authors.forename: Shunnosuke
authors.initials: S
authors.lastname: Abe
authors.forename: Eugene Hayato
authors.initials: EH
authors.lastname: Morita
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D019906:Nuclear Magnetic Resonance, Biomolecular; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D046940:Synechococcus / Q000737:chemistry / Q000235:genetics*; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D015854:Up-Regulation
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D014157:Transcription Factors