Role of disulfide bond isomerase DsbC, calcium ions, and hemin in cell-free protein synthesis of active manganese peroxidase isolated from Phanerochaete chrysosporium

Ninomiya, Ryoko; Zhu, Bo; Kojima, Takaaki; Iwasaki, Yoshinobu; Nakano, Hideo

doi:10.1016/j.jbiosc.2013.11.003

Cited by 20 publications

(14 citation statements)

References 40 publications

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“…The pTXB1 vector promotes the expression of a fusion protein rMnP1-intein-CBD, where the chitin binding domain (CBD) allowed the efficient purification of rMnP1 by a single-step affinity chromatography. Conveniently, the expression host, E. coli T7 shuffle, constitutively expresses the disulfide bond isomerase (DsbC), which has been proven to positively impact the production of recombinant MnP [28]. Additionally, the cleavage buffer contains dithiothreitol (DTT), which promotes the self-excision of the target protein.…”

Section: Discussionmentioning

confidence: 99%

“…The incorporation of physical disruption methods, such as sonication and vortex, was insufficient to disaggregate inclusion bodies; consequently, the solubilisation of inclusion bodies was achieved with 4 M urea ( Figure 5). Another point to consider is the proper refolding of MnP after recovering from inclusion bodies [16,17,28,35]. Currently there is no single method of refolding that fits for all proteins.…”

Section: Discussionmentioning

confidence: 99%

“…Such modifications included the immobilization of fungal cells [11], incubation at different ranges of temperature or pH, addition of Tween 80 or cofactors to the culture media, nitrogen limitation, growth on solid media instead of liquid cultures [12][13][14][15], and so on. Moreover, heterologous expression of MnP has been achieved in Escherichia coli [16][17][18], using the baculovirus expression system [19], in Pichia pastoris [20][21][22][23][24], in Aspergillus [8,25,26], and in a cell free system [27,28].…”

Section: Introductionmentioning

confidence: 99%

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Functional Expression and One-Step Protein Purification of Manganese Peroxidase 1 (rMnP1) from Phanerochaete chrysosporium Using the E. coli-Expression System

Pech-Canul

Carrillo-Campos

Ballinas-Casarrubias

et al. 2020

IJMS

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Manganese peroxidases (MnP) from the white-rot fungi Phanerochaete chrysosporium catalyse the oxidation of Mn2+ to Mn3+, a strong oxidizer able to oxidize a wide variety of organic compounds. Different approaches have been used to unravel the enzymatic properties and potential applications of MnP. However, these efforts have been hampered by the limited production of native MnP by fungi. Heterologous expression of MnP has been achieved in both eukaryotic and prokaryotic expression systems, although with limited production and many disadvantages in the process. Here we described a novel molecular approach for the expression and purification of manganese peroxidase isoform 1 (MnP1) from P. chrysosporium using an E. coli-expression system. The proposed strategy involved the codon optimization and chemical synthesis of the MnP1 gene for optimised expression in the E. coli T7 shuffle host. Recombinant MnP1 (rMnP1) was expressed as a fusion protein, which was recovered from solubilised inclusion bodies. rMnP1 was purified from the fusion protein using intein-based protein purification techniques and a one-step affinity chromatography. The designated strategy allowed production of an active enzyme able to oxidize guaiacol or Mn2+.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Functional Expression and One-Step Protein Purification of Manganese Peroxidase 1 (rMnP1) from Phanerochaete chrysosporium Using the E. coli-Expression System

Pech-Canul

Carrillo-Campos

Ballinas-Casarrubias

et al. 2020

IJMS

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“…Heme is a non-covalently bound prosthetic group in MnP; hence, adding exogenous hemin to the cell lysates is necessary to yield an active holoenzyme. Additionally, Ca 2+ supplementation is crucial to achieve the correct conformation, as well as the stability, of recombinant MnP [44, 45], because both Ca 2+ ions are easily lost in a thermal or alkaline environment [6, 46]. …”

Section: Discussionmentioning

confidence: 99%

Expression of a fungal manganese peroxidase in Escherichia coli: a comparison between the soluble and refolded enzymes

et al. 2016

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BackgroundManganese peroxidase (MnP) from Irpex lacteus F17 has been shown to have a strong ability to degrade recalcitrant aromatic pollutants. In this study, a recombinant MnP from I. lacteus F17 was expressed in Escherichia coli Rosetta (DE3) in the form of inclusion bodies, which were refolded to achieve an active enzyme. Further, we optimized the in vitro refolding conditions to increase the recovery yield of the recombinant protein production. Additionally, we attempted to express recombinant MnP in soluble form in E. coli, and compared its activity with that of refolded MnP.ResultsRefolded MnP was obtained by optimizing the in vitro refolding conditions, and soluble MnP was produced in the presence of four additives, TritonX-100, Tween-80, ethanol, and glycerol, through incubation at 16 °C. Hemin and Ca2+ supplementation was crucial for the activity of the recombinant protein. Compared with refolded MnP, soluble MnP showed low catalytic efficiencies for Mn2+ and H2O2 substrates, but the two enzymes had an identical, broad range substrate specificity, and the ability to decolorize azo dyes. Furthermore, their enzymatic spectral characteristics were analysed by circular dichroism (CD), electronic absorption spectrum (UV-VIS), fluorescence and Raman spectra, indicating the differences in protein conformation between soluble and refolded MnP. Subsequently, size exclusion chromatography (SEC) and dynamic light scattering (DLS) analyses demonstrated that refolded MnP was a good monomer in solution, while soluble MnP predominantly existed in the oligomeric status.ConclusionsOur results showed that two forms of recombinant MnP could be expressed in E. coli by varying the culture conditions during protein expression.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-016-0317-2) contains supplementary material, which is available to authorized users.

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“…Escherichia coli S30 extract was prepared as previously described (21). Biotinylated DNA template was prepared by PCR with Bio-EcoRI and R1 primers as shown in Fig.…”

Section: Cell-free Protein Synthesis In Emulsionmentioning

confidence: 99%

Handmade microfluidic device for biochemical applications in emulsion

Murzabaev

Kojima

Mizoguchi

et al. 2016

Journal of Bioscience and Bioengineering

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Role of disulfide bond isomerase DsbC, calcium ions, and hemin in cell-free protein synthesis of active manganese peroxidase isolated from Phanerochaete chrysosporium

Cited by 20 publications

References 40 publications

Functional Expression and One-Step Protein Purification of Manganese Peroxidase 1 (rMnP1) from Phanerochaete chrysosporium Using the E. coli-Expression System

Functional Expression and One-Step Protein Purification of Manganese Peroxidase 1 (rMnP1) from Phanerochaete chrysosporium Using the E. coli-Expression System

Expression of a fungal manganese peroxidase in Escherichia coli: a comparison between the soluble and refolded enzymes

Handmade microfluidic device for biochemical applications in emulsion

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