Dataset Information

Overexpression and Purification of Gracilariopsis chorda Carbonic Anhydrase (GcCA?3) in Nicotiana benthamiana, and Its Immobilization and Use in CO₂ Hydration Reactions.

ABSTRACT: Carbonic anhydrase (CA; EC 4.2.2.1) is a Zn-binding metalloenzyme that catalyzes the reversible hydration of CO₂. Recently, CAs have gained a great deal of attention as biocatalysts for capturing CO₂ from industrial flue gases owing to their extremely fast reaction rates and simple reaction mechanism. However, their general application for this purpose requires improvements to stability at high temperature and under in vitro conditions, and reductions in production and scale-up costs. In the present study, we developed a strategy for producing GcCA?3, a CA isoform from the red alga Gracilariopsis chorda, in Nicotiana benthamiana. To achieve high-level expression and facile purification of GcCA?3, we designed various constructs by incorporating various domains such as translation-enhancing M domain, SUMO domain and cellulose-binding domain CBM3. Of these constructs, MC-GcCA?3 that had the M and CBM3 domains was expressed at high levels in N. benthamiana via agroinfiltration with a yield of 1.0 g/kg fresh weight. The recombinant protein was targeted to the endoplasmic reticulum (ER) for high-level accumulation in plants. Specific and tight CBM3-mediated binding of recombinant GcCA?3 proteins to microcrystalline cellulose beads served as a means for both protein purification from total plant extracts and protein immobilization to a solid surface for increased stability, facilitating multiple rounds of use in CO₂ hydration reactions.

SUBMITTER: Razzak MA

PROVIDER: S-EPMC7717956 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Publications

Overexpression and Purification of Gracilariopsis chorda Carbonic Anhydrase (GcCAα3) in Nicotiana benthamiana, and Its Immobilization and Use in CO2 Hydration Reactions.

Razzak Md Abdur MA Lee Dong Wook DW Lee Junho J Hwang Inhwan I

Frontiers in plant science 20201119

Carbonic anhydrase (CA; EC 4.2.2.1) is a Zn-binding metalloenzyme that catalyzes the reversible hydration of CO2. Recently, CAs have gained a great deal of attention as biocatalysts for capturing CO2 from industrial flue gases owing to their extremely fast reaction rates and simple reaction mechanism. However, their general application for this purpose requires improvements to stability at high temperature and under in vitro conditions, and reductions in production a ...[more]

PMID: 33329625

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Project description:α1-Microglobulin (A1M) is a small glycoprotein that belongs to the lipocalin protein family. A major biological role of A1M is to protect cells and tissues against oxidative damage by clearing free heme and reactive oxygen species. Because of this, the protein has attracted great interest as a potential pharmaceutical candidate for treatment of acute kidney injury and preeclampsia. The aim of this study was to explore the possibility of expressing human A1M in plants through transient gene expression, as an alternative or complement to other expression systems. E. coli, insect and mammalian cell culture have previously been used for recombinant A1M (rA1M) or A1M production, but these systems have various drawbacks, including additional complication and expense in refolding for E. coli, while insect produced rA1M is heavily modified with chromophores and mammalian cell culture has been used only in analytical scale. For that purpose, we have used a viral vector (pJL-TRBO) delivered by Agrobacterium for expression of three modified A1M gene variants in the leaves of N. benthamiana. The results showed that these modified rA1M protein variants, A1M-NB1, A1M-NB2 and A1M-NB3, targeted to the cytosol, ER and extracellular space, respectively, were successfully expressed in the leaves, which was confirmed by SDS-PAGE and Western blot analysis. The cytosol accumulated A1M-NB1 was selected for further analysis, as it appeared to have a higher yield than the other variants, and was purified with a yield of ca. 50 mg/kg leaf. The purified protein had the expected structural and functional properties, displaying heme-binding capacity and capacity of protecting red blood cells against stress-induced cell death. The protein also carried bound chromophores, a characteristic feature of A1M and an indicator of a capacity to bind small molecules. The study showed that expression of the functional protein in N. benthamiana may be an attractive alternative for production of rA1M for pharmaceutical purposes and a basis for future research on A1M structure and function.

Dataset Information

Overexpression and Purification of Gracilariopsis chorda Carbonic Anhydrase (GcCA?3) in Nicotiana benthamiana, and Its Immobilization and Use in CO₂ Hydration Reactions.

Publications

Overexpression and Purification of <i>Gracilariopsis chorda</i> Carbonic Anhydrase (GcCAα3) in <i>Nicotiana benthamiana</i>, and Its Immobilization and Use in CO<sub>2</sub> Hydration Reactions.

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Dataset Information

Overexpression and Purification of Gracilariopsis chorda Carbonic Anhydrase (GcCA?3) in Nicotiana benthamiana, and Its Immobilization and Use in CO2 Hydration Reactions.

Publications

Overexpression and Purification of <i>Gracilariopsis chorda</i> Carbonic Anhydrase (GcCAα3) in <i>Nicotiana benthamiana</i>, and Its Immobilization and Use in CO<sub>2</sub> Hydration Reactions.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Overexpression and Purification of Gracilariopsis chorda Carbonic Anhydrase (GcCA?3) in Nicotiana benthamiana, and Its Immobilization and Use in CO₂ Hydration Reactions.