High solubility supports efficient refolding of thermally unfolded β-lactamase

Arakawa, Tsutomu; Tokunaga, Hiroko; Yamaguchi, Rui; Tokunaga, Masao

doi:10.1016/j.ijbiomac.2010.09.009

Cited by 5 publications

(7 citation statements)

References 12 publications

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“…Thus, 2 M NaCl is insufficient to cause aggregation of the heat-denatured structure, but is instead effective in charge screening and accelerates refolding. ␤-Lactamase from moderate halophile is a highly acidic protein, with the calculated pI of 4.2, and shows highly efficient refolding after heat-denaturation, though only in low salt conditions [9,37]. It forms aggregation in the presence of 2 M NaCl.…”

Section: Discussionmentioning

confidence: 99%

“…6, bottom), which has more neutral (white color) and basic (blue color) regions with some acidic regions. Highly negative-charged protein surface is one of the features of halophilic proteins [3,[8][9][10].…”

Section: Three-dimensional Structure Modeling and Electrostatic Potenmentioning

confidence: 99%

“…We have been studying the distinct characteristics of halophilic proteins, including nucleoside diphosphate kinases [4][5][6][7], ␤-lactamase [8,9] and ␣-amylase secreted from Kocuria varians [10]. The most striking features of halophilic proteins, especially the secretory proteins present in the periplasmic space or the culture medium of moderate halophiles, are their high aqueous solubility in both the native and denatured structures and the high reversibility of refolding from the denatured states without forming aggregation [6,8].…”

Section: Introductionmentioning

confidence: 99%

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Halophilic characterization of starch-binding domain from Kocuria varians α-amylase

Yamaguchi

Inoue

Tokunaga

et al. 2012

International Journal of Biological Macromolecules

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Section: Discussionmentioning

confidence: 99%

Section: Three-dimensional Structure Modeling and Electrostatic Potenmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Halophilic characterization of starch-binding domain from Kocuria varians α-amylase

Yamaguchi

Inoue

Tokunaga

et al. 2012

International Journal of Biological Macromolecules

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“…Both proteins are extremely acidic, as indicated by a high ratio of acidic/basic amino acid residues of 3.7 for HP and 2.1 for BLA. Thus, both proteins possess large net negative charges at neutral pH, which are considered to be associated with their high aqueous solubility in both native and denatured states . These proteins are strongly resistant to aggregation, suggesting that they might not form fibril structures.…”

Section: Resultsmentioning

confidence: 99%

“…Namely, halophilic proteins are characterized as acidic, low‐p I proteins. Abundant negative charges, combined with weak hydrophobicity, at neutral pH made halophilic proteins highly soluble even in unfolded structure caused by high temperature . Based on such high solubility, it may be anticipated that halophilic proteins show rather low tendency to form amyloid fibrils.…”

Section: Introductionmentioning

confidence: 99%

Amyloid fibril formation in vitro from halophilic metal binding protein: Its high solubility and reversibility minimized formation of amorphous protein aggregations

et al. 2013

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Halophilic proteins are characterized by high net negative charges and relatively small fraction of hydrophobic amino acids, rendering them aggregation resistant. These properties are also shared by histidine-rich metal binding protein (HP) from moderate halophile, Chromohalobacter salexigens, used in this study. Here, we examined how halophilic proteins form amyloid fibrils in vitro. His-tagged HP, incubated at pH 2.0 and 58 C, readily formed amyloid fibrils, as observed by thioflavin fluorescence, CD spectra, and transmission or atomic force microscopies. Under these low-pH harsh conditions, however, His-HP was promptly hydrolyzed to smaller peptides most likely responsible for rapid formation of amyloid fibril. Three major acid-hydrolyzed peptides were isolated from fibrils and turned out to readily form fibrils. The synthetic peptides predicted to form fibrils in these peptide sequences by Waltz software also formed fibrils. Amyloid fibril was also readily formed from full-length His-HP when incubated with 10-20% 2,2,2-trifluoroethanol at pH 7.8 and 25 C without peptide bond cleavage.

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Secretory production of single-chain antibody (scFv) in Brevibacillus choshinensis using novel fusion partner

Tokunaga

Mizukami²,

Yamasaki

et al. 2013

Appl Microbiol Biotechnol

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Halophilic β-lactamase (BLA) has been successfully used as a novel fusion partner for soluble expression of aggregation-prone foreign proteins in Escherichia coli cytoplasm (Appl Microbiol Biotechnol 86:649-658, 2010b). This halophilic BLA fusion technology was applied here for secretory expression in Brevibacillus. The "Brevibacillus in vivo cloning" method, recently developed by Higeta Shoyu group, for the construction and transformation of Brevibacillus expression vectors facilitates efficient screening of the production conditions of Brevibacillus expression system. Two single-chain antibodies (scFv), HyHEL-10 single chain scFv (scFvHEL) and anti-fluorescein single chain scFv (scFvFLU), were successfully secreted to culture supernatant as a fusion protein with halophilic BLA. The scFvHEL-His, purified after cleavage of BLA portion with thrombin, was fully active: it formed a stoichiometric complex with the antigen, lysozyme, and inhibited the enzymatic activity. The scFvFLU-His, similarly expressed and purified, stoichiometrically inhibited fluorescence intensity of fluorescein. The molecular mass of scFvHEL-His was determined to be 27,800 Da by light scattering measurements, indicating its monomeric structure in solution.

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High solubility supports efficient refolding of thermally unfolded β-lactamase

Cited by 5 publications

References 12 publications

Halophilic characterization of starch-binding domain from Kocuria varians α-amylase

Halophilic characterization of starch-binding domain from Kocuria varians α-amylase

Amyloid fibril formation in vitro from halophilic metal binding protein: Its high solubility and reversibility minimized formation of amorphous protein aggregations

Secretory production of single-chain antibody (scFv) in Brevibacillus choshinensis using novel fusion partner

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