Sequencing, expression and properties of triosephosphate isomerase from Entamoeba histolytica.
We have isolated a cDNA clone of the glycolytic enzyme, triosephosphate isomerase (TPI) from Entamoeba histolytica. Degenerate oligonucleotides obtained by reverse translation of conserved polypeptide sequences, derived from TPIs of other organisms, were used to amplify a 450-bp fragment using E. histolytica cDNA as a template. The fragment was used to screen a cDNA library. The isolated cDNA, encoding a protein of 261 amino acids, shares 43-52.6% positional identity with other known protozoan TPIs. The catalytic residues were conserved; nevertheless, several indels occurred at other regions in the protein sequence. The complete coding sequence of the E. histolytica TPI gene was cloned into the expression vector pRSET and expressed as a wild-type TPI enzyme (E. histolytica TPI) and as a fusion protein with an N-terminal tail of six histidine residues E. histolytica TPI-His6); both recombinant proteins were purified. Molecular modeling of E. histolytica TPI showed an identical topology to the known structures of other TPI molecules, but with a remarkable feature; more than 10 inserted residues are located in the same region of the molecular surface. Studies were performed to detect possible changes that might be caused by the inserted amino acids. The catalytic activity and oligomeric state of the purified protein were similar to that reported for TPI from other sources. In contrast, stability towards dilution, as well as thermal inactivation and unfolding assays, showed that E. histolytica TPI is significantly more stable towards denaturation than Trypanosoma brucei TPI.
Landa
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Departamento de Microbiología y Parasitología, Facultad de Medicina, Universidad Nacional Autónoma de México, México, D.F.
Rojo-Domínguez
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Jiménez
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Fernández-Velasco
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D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D004748:Entamoeba histolytica / Q000201:enzymology*; D004795:Enzyme Stability; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017510:Protein Folding; D014305:Triose-Phosphate Isomerase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification
D014305:Triose-Phosphate Isomerase