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Expression and purification of recombinant arginine decarboxylase (speA) from Escherichia coli

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Abstract

The crystal structures of almost all the enzymes of arginine metabolism have been determined, but arginine decarboxylase’s structure is not resolved yet. In order to characterize and crystallize arginine decarboxylase, we overexpressed biosynthetic arginine decarboxylase (ADC; EC 4.1.1.19, encoded by the speA gene) from Escherichia coli in the T7 expression system as a cleavable poly-His-tagged fusion construct. The expressed recombinant His10-ADC (77.3 kDa) was first purified by Ni–NTA affinity chromatography, then proteolytically digested with Tobacco Etch Virus (TEV) protease to remove the poly-His fusion tag, and finally purified by anion exchange chromatography. The His10 tag removed recombinant ADC (74.1 kDa)’s typical yield was 90 mg from 1 l of culture medium with purity above 98%. The recombinant ADC was assayed for decarboxylase activity, showing decarboxylase activity of 2.8 U/mg, similar to the purified native E. coli ADC. The decarboxylase activity assay also showed that the purified recombinant ADC tolerated broad ranges of pH (pH 6–9) and temperature (20–80°C). Our research may facilitate further studies of ADC structure and function, including the determination of its crystal structure by X-ray diffraction.

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Abbreviations

ADC:

Arginine decarboxylase

BCA:

Bicinchoninic acid

DTT:

Dithiothreitol

EDTA:

Ethylenediaminetetraacetic acid

IPTG:

Isopropyl β-D-thiogalactopyranoside

Ni-NTA:

Nickel-nitrilotriacetic acid

PLP:

Pyridoxal phosphate

SDS-PAGE:

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

TEV protease:

Tobacco Etch Virus protease

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Acknowledgments

We especially thank Professor Zhihong Zhang for the critical reviews, additions, and useful suggestions on this manuscript drafts. The work was supported by the grants (No. 30600107, 30500113 and 30670499) from the National Natural Science Foundation of China and Shanghai Leading Academic Discipline Project (Project number B111). Rui Liu, Xudong Wu and Di Wu were supported by the National Talent Training Fund in Basic Research of China (No. J0630643) and Xi Yuan Scholar Program (2008).

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  1. Department of Physiology and Biophysics, School of Life Sciences, Fudan University, 200433, Shanghai, China

    Jiaping Song, Chuanwen Zhou, Rui Liu, Xudong Wu, Di Wu, Xiaojian Hu & Yu Ding

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  1. Jiaping Song
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  2. Chuanwen Zhou
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  3. Rui Liu
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  4. Xudong Wu
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  5. Di Wu
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  6. Xiaojian Hu
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  7. Yu Ding
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Correspondence to Yu Ding.

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Song, J., Zhou, C., Liu, R. et al. Expression and purification of recombinant arginine decarboxylase (speA) from Escherichia coli . Mol Biol Rep 37, 1823–1829 (2010). https://doi.org/10.1007/s11033-009-9617-0

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  • DOI: https://doi.org/10.1007/s11033-009-9617-0

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