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Renaturation, activation, and practical use of recombinant duplex-specific nuclease from Kamchatka crab

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Abstract

We overexpressed duplex-specific nuclease (DSN) from Kamchatka crab in Escherichia coli cells and developed procedures for purification, renaturation, and activation of this protein. We demonstrated identity of the properties of the native and recombinant DSN. We also successfully applied the recombinant DSN for full-length cDNA library normalization.

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Abbreviations

BCIP:

5-bromo-4-chloro-3-indolyl phosphate

Cys:

cysteine

Cys-Cys:

cystine

DSN:

duplex-specific nuclease

GSH:

reduced glutathione

GSSG:

oxidized glutathione

IPTG:

isopropyl β-D-thiogalactopyranoside

LB:

Luria-Bertani medium

NBT:

nitro blue tetrazolium

PBS:

phosphate buffered saline

PDI:

protein disulfide isomerase

PEG:

polyethylene glycol

SDS:

sodium dodecyl sulfate

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Authors and Affiliations

  1. Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, 117997, Moscow, Russia

    V. E. Anisimova, D. V. Rebrikov, P. A. Zhulidov, D. B. Staroverov, S. A. Lukyanov & A. S. Shcheglov

Authors
  1. V. E. Anisimova
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  2. D. V. Rebrikov
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  3. P. A. Zhulidov
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  4. D. B. Staroverov
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  5. S. A. Lukyanov
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  6. A. S. Shcheglov
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Additional information

Original Russian Text © V. E. Anisimova, D. V. Rebrikov, P. A. Zhulidov, D. B. Staroverov, S. A. Lukyanov, A. S. Shcheglov, 2006, published in Biokhimiya, 2006, Vol. 71, No. 5, pp. 636–644.

Originally published in Biochemistry (Moscow) On-Line Papers in Press, as Manuscript BM05-241, April 9, 2006.

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Anisimova, V.E., Rebrikov, D.V., Zhulidov, P.A. et al. Renaturation, activation, and practical use of recombinant duplex-specific nuclease from Kamchatka crab. Biochemistry (Moscow) 71, 513–519 (2006). https://doi.org/10.1134/S0006297906050075

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  • DOI: https://doi.org/10.1134/S0006297906050075

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