Cloning and Expression of Malarial Pyrimidine Enzymes
Autor: | R. I. Menz, Richard I. Christopherson, Maryam Shojaei, D. Kuehn, Olivier Cinquin |
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Rok vydání: | 2004 |
Předmět: |
Orotate Phosphoribosyltransferase
Genetic Vectors Orotidine-5'-Phosphate Decarboxylase Plasmodium falciparum medicine.disease_cause Biochemistry Catalysis law.invention Open Reading Frames Viral Proteins Plasmid law Escherichia coli Genetics medicine Animals Cloning Molecular Codon Gene Dihydroorotase DNA Primers chemistry.chemical_classification DNA-Directed RNA Polymerases N-Acetylmuramoyl-L-alanine Amidase General Medicine Molecular biology Recombinant Proteins Malaria Protein Structure Tertiary Enzyme Models Chemical chemistry Recombinant DNA Molecular Medicine Orotate phosphoribosyltransferase Hybrid plasmid Plasmids |
Zdroj: | Nucleosides, Nucleotides and Nucleic Acids. 23:1459-1465 |
ISSN: | 1532-2335 1525-7770 |
Popis: | We have cloned genes encoding three enzymes of the de novo pyrimidine pathway using genomic DNA from Plasmodium falciparum and sequence information from the Malarial Genome Project. Genes encoding dihydroorotase (reaction 3), orotate phosphoribosyltransferase (reaction 5), and OMP decarboxylase (reaction 6) have been cloned into the plasmid pET 3a or 3d with a thrombin cleavable 9xHis tag at the C-terminus and the enzymes were expressed in Escherichia coli. To overcome the toxicity of malarial OMP decarboxylase when expressed in E. coli, and the unusual codon usage of the malarial gene, a hybrid plasmid, pMICO, was constructed which expresses low levels of T7 lysozyme to inhibit T7 RNA polymerase used for recombinant expression, and extra copies of rare tRNAs. Catalytically-active OMP decarboxylase has been purified in tens of milligrams by chromatography on Ni-NTA. The gene encoding orotate phosphoribosyltransferase includes an extension of 66 amino acids from the N-terminus when compared with sequences for this enzyme from other organisms. We have found that other pyrimidine enzymes also contain unusual protein inserts. Milligram quantities of pure recombinant malarial enzymes from the pyrimidine pathway will provide targets for development of novel antimalarial drugs. |
Databáze: | OpenAIRE |
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