Cinquin Lab

Christopherson R.I., Cinquin O., Shojaei M., Kuehn D., Menz R.I., Nucleos. Nucleot. Nucl. 23(8-9), pp1459-1465 (2004)

Abstract

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.

Menz R.I., Cinquin O., Christopherson R.I. Ann. Trop. Med. Parasitol. 96(5), pp469-476 (2002)

Abstract

The coding region of a putative orotidine 5′-monophosphate decarboxylase gene from Plasmodium falciparum was identified in genomic data from the Malarial Genome Sequencing Project. The gene encodes a protein of 323 amino acids with a predicted molecular weight of 37.8 kDa. The gene was cloned into a bacterial expression vector and over-expressed in Escherichia coli. The recombinant protein was purified and shown to have orotidine 5′-monophosphate decarboxylase activity, confirming the identity of the gene.

Cinquin O., Christopherson R.I., Menz R.I., Mol. Biochem. Parasitol. 117(2), pp245-247 (2001)

Abstract

The RIG plasmid [Baca and Hol, 2000, Int. J. Parasitol. 30, 113-118] encodes tRNA genes rare in Escherichia coli, enabling codon-biased Plasmodium genes to be over-expressed in E. coli. Expression of orotidine-5′-monophosphate decarboxylase (EC 4.1.1.23) from Plasmodium falciparum was found to be toxic to E. coli BL21(DE3) cells carrying the RIG plasmid. Unfortunately, the RIG plasmid is incompatible with the pLysS plasmid commonly used with pET expression vectors to allow over-expression of recombinant proteins toxic to E. coli by repressing their basal level expression. We describe here a new hybrid plasmid carrying the gene for T7 lysozyme and those for the rare E. coli codons argU, ileX and glyT, which facilitates over-expression of orotidine-5′-monophosphate decarboxylase (EC 4.1.1.23) from P. falciparum.