Cloning and Expression of a Prokaryotic Enzyme, Arginine Deiminase, from a Primitive Eukaryote Giardia intestinalis

Philip J. Schofield,Eric O. Sekyere,Michael R. Edwards,Leigh A. Knodler,Thomas S. Stewart

doi:10.1074/jbc.273.8.4470

Abstract

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 in Escherichia coli and purified to homogeneity. The recombinant protein was found to have characteristics comparable with those of the native enzyme.

Highlights

Giardia intestinalis is one of the most commonly transmitted intestinal pathogens in the world
Amplification of a Fragment of the Arginine Deiminase Gene—Oligonucleotide primers designed from tryptic peptides of the purified arginine deiminase were used in PCR to amplify a portion of the target gene
We have previously demonstrated that the arginine transport system in Giardia is supportive of its position as a transition between prokaryotes and eukaryotes, and the metabolism of arginine reflects the primitive nature of this parasite

Summary

EXPERIMENTAL PROCEDURES

Cell Culture Conditions—G. intestinalis trophozoites, Portland I strain, were cultured as described previously [11]. Nucleic Acid Hybridizations—Trophozoite genomic DNA (10 ␮g) was digested to completion with either BamHI, EcoRI, HindIII, or PstI, and the resulting fragments were separated by electrophoresis on 1% (w/v) agarose gels. The RNA was transferred to Zeta-Probe blotting membrane (Bio-Rad) and hybridized overnight at 60 °C with a randomly labeled (MegaPrime, Amersham Corp.) 1.5-kb HindIII/PstI fragment from the arginine deiminase gene. Colonies were transferred to Hybond Nϩ membrane and hybridized overnight at 60 °C with the 600-bp amplicon that had been randomly labeled (MegaPrime, Amersham Corp.) This procedure was repeated with HindIII-restricted fragments of approximately 3.1 kb. Expression of the Recombinant Protein—A 1.8-kb KpnI/BamHI fragment representing the entire arginine deiminase coding sequence was amplified by PCR from Giardia genomic DNA using Pfu polymerase (Boehringer Mannheim).

RESULTS

Nucleotide sequence

Crude extract

Guanidino group altered or absent Lysine Ornithine

DISCUSSION