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Tokunaga H.,Kagoshima University | Arakawa T.,Alliance Protein Laboratory | Tokunaga M.,Kagoshima University
Protein and Peptide Letters | Year: 2013

One of the hallmarks of halophilic properties is reversibility of thermal unfolding. A nucleoside diphosphate kinase (NDK) from a moderate halophile Halomonas sp. 593 (HaNDK) follows this behavior. His-tagged chimeric NDK (HisPaHaNDK) consisting of an N-terminal half of a non-halophilic Pseuodomonas aeruginosa NDK (PaNDK) and a Cterminal half of HaNDK loses this reversible property, indicating a critical role of the N-terminal portion of PaNDK in determining the reversibility of the chimeric protein. Various mutations were introduced at Arg45 and Lys61, based on the model NDK structure. It appears that having Glu at position 45 is critical in conferring the thermal reversibility to HisPa-HaNDK chimeric protein. © 2013 Bentham Science Publishers. Source


Yamaguchi R.,Kagoshima University | Inoue Y.,Kagoshima University | Tokunaga H.,Kagoshima University | Ishibashi M.,Kagoshima University | And 4 more authors.
International Journal of Biological Macromolecules | Year: 2012

The tandem starch-binding domains (KvSBD) located at carboxy-terminal region of halophilic α-amylase from moderate halophile, Kocuria varians, were expressed in E. coli with amino-terminal hexa-His-tag and purified to homogeneity. The recombinant KvSBD showed binding activity to raw starch granules at low to high salt concentrations. The binding activity of KvSBD to starch was fully reversible after heat-treatment at 85 °C. Circular dichroism and thermal scanning experiments indicated that KvSBD showed fully reversible refolding upon cooling after complete melting at 70 °C in the presence of 0.2-2.0. M NaCl. The refolding rate was enhanced with higher salt concentration. © 2011 Elsevier B.V. Source


Yamaguchi R.,Kagoshima University | Arakawa T.,Alliance Protein Laboratory | Tokunaga H.,Kagoshima University | Ishibashi M.,Kagoshima University | Tokunaga M.,Kagoshima University
Protein and Peptide Letters | Year: 2012

The starch binding domain of α-amlylase from moderate halophile was expressed in E. coli with His tag (His- SBD12) and characterized for its halophilic properties. His-SBD12 was stable up to 35 °C and showed binding activity, although at reduced level, to amylose even in the absence of NaCl. Both NaCl and specific ligands exhibited insignificant influence on the secondary structure of His-SBD12, but showed significant stabilization effects against thermal unfolding concentration-dependently, showing its halophilic properties. NaCl increased thermal stability of His-SBD12 by 4 °C at 0.2 M and 15 °C at 2 M, and enhanced refolding rate by ∼7-fold at 0.2 M and ∼170-fold at 2 M. Its specific ligands, β- cyclodextrin (at 3 mM) and maltose (at 470 mM), also stabilized the protein by 11 °C, most likely reflecting affinity difference between these two ligands. However, they showed marginal effects on refolding rate. These observations suggest that although binding of NaCl and specific ligands to the native structure can explain their stabilization effects on His- SBD12, it is not a sole factor for modulating their effects on folding of His-SBD12. © 2012 Bentham Science Publishers. Source


Tokunaga H.,Kagoshima University | Furukawa M.,Kagoshima University | Arakawa T.,Alliance Protein Laboratory | Tokunaga M.,Kagoshima University
International Journal of Biological Macromolecules | Year: 2013

We have previously found that the N-terminal sequence of the outer membrane protein from moderate halophile is similar to the sequence of the well-known pore forming porin proteins from other Gram-negative bacteria. This highly expressed outer membrane protein was purified from Halomonas sp. 40 and reconstituted into liposome. It showed a permeability activity in the liposome swelling assay. Based on the N-terminal and internal amino acid sequences of this major outer membrane, we have cloned here the porin gene, hopP (halophilic outer membrane protein), from Halomonas sp. 40. The hopP gene encodes the porin precursor comprising 366 amino acid residues that include a 21 amino acid signal peptide. Mature porin (345 amino acids, 37,611. Da) is a highly acidic protein, just as is so for many halophilic proteins and was soluble when expressed in Escherichia coli with N-terminal His-tag. Purified recombinant His-porin was soluble even after heat-treatment at 95 °C for 5. min in the absence of salt. Circular dichroism analysis of His-porin showed conversion into a β-sheet rich structure by the addition of NaCl at 0.9-2.7. M. © 2012 Elsevier B.V. Source


Yamaguchi R.,Kagoshima University | Arakawa T.,Alliance Protein Laboratory | Tokunaga H.,Kagoshima University | Ishibashi M.,Kagoshima University | Tokunaga M.,Kagoshima University
Protein Journal | Year: 2012

Periplasmic metal binding protein characterized by high histidine content was cloned from moderate halophile, Chromohalobacter salexigens. The protein, termed histidine-rich metal binding protein (HP), was expressed in and purified from E. coli as a native form. HP bound to Ni- and Cu-loaded chelate columns with high affinity, and Co- and Zn-columns with moderate affinity. Although the secondary structure was not grossly altered by the addition of 0.2-2.0 M NaCl, the thermal transition pattern was considerably shifted to higher temperature with increasing salt concentration: melting temperature was raised by ∼ 20°C at 2.0 M NaCl over the melting temperature at 0.2 M NaCl. HP showed reversible refolding from thermal melting in 0.2-1.15 M NaCl, while it formed irreversible aggregates upon thermal melting at 2 M NaCl. Addition of 0.01-0.1 mM NiSO 4 stabilized HP against thermal melting with high reversibility, while addition above 0.5 mM resulted in irreversible melting due to aggregation. © Springer Science+Business Media, LLC 2011. Source

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