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Expression, purification and preliminary characterization of mussel (Mytilus galloprovincialis) metallothionein MT20

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Abstract

Metallothioneins are rather ubiquitous metal-binding proteins induced by stressing or physiological stimuli. Two major metallothionein isoforms have been identified in mussel: MT10 and MT20. Nevertheless the high sequence homology, the two isoforms exhibit different expression and inducibility in vivo. We cloned and produced in Escherichia coli the MT20 isoform from Mytilus galloprovincilis. cDNA was subcloned into pGEX-6P.1 vector, in frame with a sequence encoding a glutathione-S-transferase (GST) tail. Recombinant protein was purified to electrophoretic homogeneity by affinity chromatography. After enzymatic cleavage of the GST tail the MT moiety was recovered with a final yield of about 5 mg of protein per litre of bacterial culture. The metal-binding ability of MT20 was assessed by absorption spectroscopy upon addition of cadmium equivalents and the metal release was checked as a function of the environment pH. Moreover the protein was analysed for the propensity to polymerization, typical of this class of protein, before and after exposure to reducing and alkylating agents.

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Abbreviations

DTNB:

5′,5′-dithio-bis(2-nitrobenzoic acid)

DTT:

1,4 dithio-dl-threitol

GST:

glutathione S-transferase

IA:

iodoacetamide

IPTG:

isopropyl-β-d-thiogalactopyranoside

MT:

metallothionein

NEM:

N-ethylmaleimide

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Acknowledgments

We would like to extend our gratitude to Prof. Gabriella Gallo for her scientific collaboration, to Drs. Cristina Borghi and Cristina Lanza for the technical assistance. This research was supported by the University of Genova Project for year 2002. This work was also granted by the 5th UE Framework Program project Biological Effects of Environmental Pollution in marine coastal ecosystems (BEEP). (Contract N°.EVK3–2000–00543).

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Authors and Affiliations

  1. Department of Biophysical Sciences and Technologies M. & O (Di.S.T.Bi.M.O.), University of Genova, Largo Rosanna Benzi 10, 16132, Genova, Italy

    Myriam Grattarola, Mara Carloni & Laura Vergani

  2. Department of Environmental and Life Sciences, University of Piemonte Orientale, Via Bellini 25 G, 15100, Alessandria, Italy

    Francesco Dondero & Aldo Viarengo

  3. Center on Biology and Chemistry of Trace Metals, University of Genova, Corso Europa 26, 16132, Genova, Italy

    Aldo Viarengo

Authors
  1. Myriam Grattarola
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  2. Mara Carloni
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  3. Francesco Dondero
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  4. Aldo Viarengo
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  5. Laura Vergani
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Correspondence to Laura Vergani.

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Grattarola, M., Carloni, M., Dondero, F. et al. Expression, purification and preliminary characterization of mussel (Mytilus galloprovincialis) metallothionein MT20. Mol Biol Rep 33, 265–272 (2006). https://doi.org/10.1007/s11033-006-9009-7

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  • DOI: https://doi.org/10.1007/s11033-006-9009-7

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