Cloning and expression in Escherichia coli of a hypoxanthine-guanine phosphoribosyltransferase-encoding cDNA from Plasmodium falciparum.
The enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT) of Plasmodium falciparum plays a key role in the salvage of preformed purine nucleotides from parasite-infected erythrocytes. Since P. falciparum cannot synthesize purines de novo, development of inhibitors specific for the parasite HGPRT should be an effective method of chemotherapy. To provide sufficient amounts of HGPRT for biochemical and crystallographic analysis, we have isolated the P. falciparum HPRT cDNA sequence and expressed it in an Escherichia coli strain deficient for both de novo purine synthesis and guanine utilization (strain GP120). GP120 cells containing the P. falciparum HPRT plasmid vector (pRD500), when grown in the presence of isopropyl-beta-D-thiogalactopyranoside (IPTG) which induces the tac promoter of the expression vector, produce a novel protein of 26 kDa, which is in agreement with the predicted Mr deduced from the HPRT cDNA open reading frame. In addition, we have demonstrated significant HGPRT activity in cell-free extracts of GP120[pRD500] cultures grown in minimal medium containing xanthine, as the sole source of purines, and IPTG.
Department of Biochemistry, Southern Research Institute, Birmingham, AL 35255-5305.
G
G
Vasanthakumar
R L
RL
Davis
M A
MA
Sullivan
J P
JP
Donahue
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics*; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics*; D008969:Molecular Sequence Data; D010957:Plasmids; D010963:Plasmodium falciparum / Q000201:enzymology / Q000235:genetics*; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid
D004247:DNA; D007041:Hypoxanthine Phosphoribosyltransferase