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Molecular cloning, sequence determination and heterologous expression of nucleoside diphosphate kinase from Pisum sativum

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Abstract

Protein sequence data derived from the N-terminal region of a 17 kDa polypeptide associated with the microsomal membrane fraction from Pisum sativum was used to design degenerate oligonucleotides which were used to amplify P. sativum cDNA via the polymerase chain reaction (PCR). Amplified cDNA was used as a probe to screen a P. sativum cDNA library and a cDNA clone, NDK-P1 was isolated and sequenced. The protein encoded by NDK-P1 had a calculated molecular mass of 16485 Da and possessed substantial homology with nucleoside diphosphate kinases (NDKs) isolated and cloned from other sources. High levels of expression of NDK-P1 protein were achieved in Escherichia coli using a T7-driven expression system. Recombinant NDK-P1 protein was shown to possess NDK activity and had similar biochemical characteristics to NDKs isolated from other sources. The Michaelis constants for a variety of nucleoside diphosphate (NDP) substrates were found to be broadly similar to those reported for other NDKs, with thymidine nucleotides being the sustrates of greatest affinity.

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Author notes

  1. P. M. Finan

    Present address: Yamanouchi Research Institute, Littlemore Hospital, OX4 4XN, Oxford, UK

Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, University of Leeds, LS2 9JT, Leeds, UK

    P. M. Finan, I. R. White, S. H. Redpath, J. B. C. Findlay & P. A. Millner

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  1. P. M. Finan
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  2. I. R. White
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  3. S. H. Redpath
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  4. J. B. C. Findlay
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  5. P. A. Millner
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Finan, P.M., White, I.R., Redpath, S.H. et al. Molecular cloning, sequence determination and heterologous expression of nucleoside diphosphate kinase from Pisum sativum . Plant Mol Biol 25, 59–67 (1994). https://doi.org/10.1007/BF00024198

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  • DOI: https://doi.org/10.1007/BF00024198

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