Cloning of the triosephosphate isomerase gene of Plasmodium falciparum and expression in Escherichia coli.
A major supply of energy in the rapidly multiplying intraerythrocytic Plasmodium falciparum is from the glycolytic pathway. We have isolated the cDNA and genomic clones of the glycolytic enzyme, triosephosphate isomerase (TPI) by polymerase chain reaction (PCR). Degenerate oligonucleotides obtained by reverse translation of conserved polypeptide sequences derived from TPIs of other organisms, were used to prime PCR on P. falciparum DNA. The P. falciparum TPI gene is interrupted by a single intron which divides the coding region into two exons. The coding region encodes a protein of 248 amino acids which is of the same size as TPIs from other organisms and shares 42-45% homology with other known eukaryotic TPIs. On comparison with human TPI the catalytic domain was found to be highly conserved, while significant variations occurred at the other regions in the protein sequence. The P. falciparum TPI gene was cloned into the expression vector pTrc99A and hyperexpressed as an unfused protein in Escherichia coli. The 28-kDa protein was shown to be catalytically active.
Ranie
J
J
Astra Research Centre India, Malleshwaram, Bangalore.
Kumar
V P
VP
Balaram
H
H
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular; D017931:DNA Primers; D016054:DNA, Protozoan / Q000302:isolation & purification / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D015870:Gene Expression; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D010963:Plasmodium falciparum / Q000201:enzymology* / Q000235:genetics*; D011061:Poly A / Q000302:isolation & purification / Q000378:metabolism; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000302:isolation & purification / Q000378:metabolism; D016053:RNA, Protozoan / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014305:Triose-Phosphate Isomerase / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification
D017931:DNA Primers; D016054:DNA, Protozoan; D012333:RNA, Messenger; D016053:RNA, Protozoan; D011994:Recombinant Proteins; D011061:Poly A; D014305:Triose-Phosphate Isomerase