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Tokunaga H.,Kagoshima University | Saito S.,Kagoshima University | Saito S.,Epoch Medical International Inc. EMI | Sakai K.,Kagoshima University | And 6 more authors.
Applied Microbiology and Biotechnology | Year: 2010

The amino acid composition of halophilic enzymes is characterized by an abundant content of acidic amino acid, which confers to the halophilic enzymes extensive negative charges at neutral pH and high aqueous solubility. This negative charge prevents protein aggregation when denatured and thereby leads to highly efficient protein refolding. β-Lactamase from periplasmic space of moderate halophile (BLA), a typical halophilic enzyme, can be readily expressed as a native, active form in Escherichia coli cytoplasm. Similar to other halophilic enzymes, BLA is soluble upon denaturation by heat or urea treatments and, hence, can be efficiently refolded. Such high solubility and refolding efficiency make BLA a potential fusion partner for expression of aggregation-prone heterologous proteins to be expressed in E. coli. Here, we succeeded in the soluble expression of several "difficult-to-express" proteins as a BLA fusion protein and verified biological activities of human interleukin 1α and human neutrophil α-defensin, HNP-1. © 2009 Springer-Verlag.

Qian X.,University of Massachusetts Amherst | Qian X.,Institute Of Bioengineering And Nanotechnology, Singapore | Mester T.,University of Massachusetts Amherst | Mester T.,University of Michigan | And 9 more authors.
Biochimica et Biophysica Acta - Bioenergetics | Year: 2011

Previous studies with Geobacter sulfurreducens have demonstrated that OmcS, an abundant c-type cytochrome that is only loosely bound to the outer surface, plays an important role in electron transfer to Fe(III) oxides as well as other extracellular electron acceptors. In order to further investigate the function of OmcS, it was purified from a strain that overproduces the protein. Purified OmcS had a molecular mass of 47 015 Da, and six low-spin bis-histidinyl hexacoordinated heme groups. Its midpoint redox potential was -212 mV. A thermal stability analysis showed that the cooperative melting of purified OmcS occurs in the range of 65-82 °C. Far UV circular dichroism spectroscopy indicated that the secondary structure of purified OmcS consists of about 10% α-helix and abundant disordered structures. Dithionite-reduced OmcS was able to transfer electrons to a variety of substrates of environmental importance including insoluble Fe(III) oxide, Mn(IV) oxide and humic substances. Stopped flow analysis revealed that the reaction rate of OmcS oxidation has a hyperbolic dependence on the concentration of the studied substrates. A ten-fold faster reaction rate with anthraquinone-2,6-disulfonate (AQDS) (25.2 s - 1) was observed as compared to that with Fe(III) citrate (2.9 s- 1). The results, coupled with previous localization and gene deletion studies, suggest that OmcS is well-suited to play an important role in extracellular electron transfer. © 2011 Elsevier B.V.

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.

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.

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.

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.

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

Kocuria varians alpha-amylase contains tandem starch-binding domains SBD1-SBD2 (SBD12) that possess typical halophilic characteristics. Recombinant tandem domains SBD12 and single domain SBD1, both with amino-terminal hexa-His tag, were expressed in and purified to homogeneity from Escherichia coli. The circular dichroism (CD) spectrum of His-SBD12 was characterized by a positive peak at 233 nm ascribed to the aromatic stacking. Although the signal occurred in the far UV region, it is an indication of tertiary structure folding. CD spectrum of single domain His-SBD1 exhibited the same peak position, signal intensity and spectral shape as those of His-SBD12, suggesting that the aromatic stacking must occur within the domain, and that two SBD domains in SBD12 and SBD1 has a similar folded structure. This structural observation was consistent with the biological activity that His-SBD1 showed binding activity against raw starch granules and amylose resin with 70-80% efficiency compared with binding of equimolar His-SBD12. Although the thermal unfolding rate of SBD12 and SBD1 were similar, the refolding rates of SBD12 and SBD1 from thermal melting were greatly different: His-SBD12 refolded slowly (T1/2 = ∼84 min), while refolding of single domain His-SBD1 was found to be 20-fold faster (T 1/2 = 4.2 min). The possible mechanism of this large difference in refolding rate was discussed. Maltose at 20 mM showed 5-6 °C increase in thermal melting of both His-SBD12 and His-SBD1, while its effects on the time course of unfolding and refolding were insignifican. © Springer Science+Business Media, LLC 2012.

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.

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