A Southern blot analysis of the Panagrellus redivivus ornithine decarboxylase (ODC) gene suggests that it is a single-copy gene that resides on a genomic 3.2 kb EcoRI fragment. Phage clones possessing ODC gene sequences were isolated from a genomic EMBL-4 library and purified. The phage DNA inserts were analysed and a 3.2 kb EcoRI fragment containing the entire ODC gene was isolated. The nucleotide sequence analysis of this fragment reveals that the gene is interrupted by two introns of 47 and 49 bp. In the 5´ non-translated region of the gene, putative AP1, VPE2 and c-Myc binding sites were identified. The ODC cDNA was expressed in a bacterial system as a His-fusion protein and the enzyme was purified by Ni2+-chelating affinity chromatography. The subunit molecular mass, as deduced from the cDNA and shown by SDS/PAGE, is 47.1 kDa. On the basis of gel filtration analyses it is shown that the active enzyme is a dimer. The specific enzyme activity was determined to be 4.2 μmol CO2/min/mg protein. The enzyme is dependent on pyridoxal 5-phosphate as a cofactor, and the presence of dithioerythritol or other thiol-reducing agents is essential for maximal activity. The Km value for l-ornithine was determined as 44 μM. The Ki values for putrescine, α-difluoromethylornithine, α-hydrazino-ornithine and α-methylornithine were calculated as 51, 34, 0.34 and 42 μM respectively.
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July 1996
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Research Article|
July 01 1996
Panagrellus redivivus ornithine decarboxylase: structure of the gene, expression in Escherichia coli and characterization of the recombinant protein
Gabriele NIEMANN;
Gabriele NIEMANN
1Bernhard Nocht Institute for Tropical Medicine, Department of Biochemistry, Bernhard-Nocht-Strasse 74, D-20359 Hamburg, Federal Republic of Germany
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Hans von BESSER;
Hans von BESSER
1Bernhard Nocht Institute for Tropical Medicine, Department of Biochemistry, Bernhard-Nocht-Strasse 74, D-20359 Hamburg, Federal Republic of Germany
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Rolf D. WALTER
Rolf D. WALTER
*
*To whom correspondence should be addressed.
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Publisher: Portland Press Ltd
Received:
February 05 1996
Accepted:
February 27 1996
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 1996
1996
Biochem J (1996) 317 (1): 135–140.
Article history
Received:
February 05 1996
Accepted:
February 27 1996
Citation
Gabriele NIEMANN, Hans von BESSER, Rolf D. WALTER; Panagrellus redivivus ornithine decarboxylase: structure of the gene, expression in Escherichia coli and characterization of the recombinant protein. Biochem J 1 July 1996; 317 (1): 135–140. doi: https://doi.org/10.1042/bj3170135
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