Cloning and expression of uridine phosphorylase gene in plasmodium falciparum

Abstract

Uridine phosphorylase in an enzyme in the family of nucleoside phosphorylase that is an important biochemical reaction in the salvage pathway of pyrimidine nucleotide. Host cell of P. falciparum can obtain uracil from salvage pathway by the activity of uridine phosphorylase and the gene encoding enzyme is identified. In contrast to host cells, P. falciparum can only obtain pyrimidines from de novo and several enzymes for salvage of pyrimidine nucleotides were not detected. However, the uridine phosphorylase was demonstrated. The purpose of this study was to identify P. falciparum uridine phosphorylase gene and to heterologously express in a bacterial system and to study on the kinetics of the enzyme. The bioinformatics underlying NCBI resources was used to identify the gene and to design primers, then the PCR product of candidate gene was cloned into E. coli. The DNA clones were subsequently to sequencing and analysis of the sequence by the BLAST program, compared with the GenBank database. We have studied on the expression of P. falciparum uridine phosphorylase gene by cloning and expression in E. coli, then purified protein is studied for kinetics of the uridine phosphorylase activity. We have found that the nucleotide sequence of candidate gene from PCR amplification is identical to the nucleotide sequence in the genome of P. falciparum in GenBank database with 99% identity by BLAST program. The purified protein from the expression in E. coli shows that it has uridine phosphorylase activity with specific activity of 342±82 nmol/min/mg protein and its kcat value is 1.18 min⁻¹