SEQUENCING, EXPRESSION AND PROPERTIES OF TRIOSEPHOSPHATE ISOMERASE FROM ENTAMOEBA-HISTOLYTICA

We have isolated a cDNA clone of the glycolytic enzyme, triosephosphat e isomerase (TPI) from Entamoeba histolytica. Degenerate oligonucleoti des 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 use d to screen a cDNA library. The isolated cDNA, encoding a protein of 2 61 amino acids: shares 43-52.6% positional identity with other known p rotozoan TPIs. The catalytic residues were conserved; nevertheless, se veral indels occurred at other regions in the protein sequence. The co mplete 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-His(6)); both recombinant pr oteins 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 loca ted in the same region of the molecular surface. Studies were performe d to detect possible changes thar might be caused by the inserted amin o acids. The catalytic activity and oligomeric state of the purified p rotein were similar to that reported for TPI from other sources. In co ntrast, stability towards dilution, as well as thermal inactivation an d unfolding assays, showed that E. histolytica TPI is significantly mo re stable towards denaturation than Trypanosoma brucei TPI.