Cloning and expression of a prokaryotic enzyme, arginine deiminase, from a primitive eukaryote Giardia intestinalis
| Autor: | Leigh A. Knodler, Thomas S. Stewart, Michael R. Edwards, Philip J. Schofield, Eric Sekyere |
|---|---|
| Rok vydání: | 1998 |
| Předmět: |
Arginine
Hydrolases Molecular Sequence Data Biology Biochemistry Peptide Mapping chemistry.chemical_compound Citrulline Animals Northern blot Amino Acid Sequence Cloning Molecular Molecular Biology Arginine deiminase Gene chemistry.chemical_classification Base Sequence Sequence Homology Amino Acid Protein arginine methyltransferase 5 Gene Amplification Chromosome Mapping Cell Biology DNA Protozoan Blotting Northern Molecular biology Amino acid Open reading frame chemistry Spectrometry Mass Matrix-Assisted Laser Desorption-Ionization Giardia lamblia |
| Zdroj: | The Journal of biological chemistry. 273(8) |
| ISSN: | 0021-9258 |
| Popis: | Arginine deiminase (EC 3.5.3.6) catalyzes the irreversible catabolism of arginine to citrulline in the arginine dihydrolase pathway. This pathway has been regarded as restricted to prokaryotic organisms but is an important source of energy to the primitive protozoan Giardia intestinalis. In this paper we report the cloning and expression of the arginine deiminase gene from this parasite. Degenerate oligonucleotides based on amino acid sequences of tryptic peptides from the purified protein were used to amplify a portion of the arginine deiminase gene. This was then used as a probe to screen HindIII and PstI “mini” libraries to obtain two overlapping clones that contained the arginine deiminase gene. The open reading frame encoded 581 amino acids including all of the tryptic peptides that were sequenced and corresponded to a molecular mass of 67 kDa. Northern blot analysis identified a single 1.8-kilobase transcript in both trophozoites and encysting cells. Arginine deiminase was successfully expressed inEscherichia coli and purified to homogeneity. The recombinant protein was found to have characteristics comparable with those of the native enzyme. |
| Databáze: | OpenAIRE |
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