The calcium binding protein of Entamoeba histolytica: expression in Escherichia coli and immunochemical characterization.
The gene encoding calcium binding protein (CaBP) of Entamoeba histolytica is single-copy and transcribes a single class of mRNA. The coding region of the gene, amplified from a genomic clone of CaBP by polymerase chain reaction, was cloned into the Escherichia coli expression vector pET-3c. The expressed protein was 15 kD in size, which agreed with the predicted size from the open reading frame. The recombinant protein showed altered mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) after binding calcium and was found to be resistant to boiling, a property shared by many calcium binding proteins. A polyclonal antibody raised against the recombinant CaBP recognized a 15 kD protein in lysates from E. histolytica pathogenic strain HM-1:IMSS. The amebic protein could compete with the recombinant protein in binding to the antibody. While the antibody recognized lysates from a number of E. histolytica strains, it failed to cross-react with other Entamoeba species, e.g., E. invadens and E. moshkovskii. Moreover, no homologue of the CaBP gene could be detected in these species by using a set of degenerate primers for amplification by polymerase chain reaction (PCR). Our data demonstrate that amebic CaBP can be expressed in E. coli and that this protein is detectable only in E. histolytica but not in other Entamoeba.
Prasad
J
J
School of Life Sciences, Jawaharlal Nehru University, New Delhi, India.
Bhattacharya
S
S
Bhattacharya
A
A
D000818:Animals; D000913:Antibodies, Protozoan / Q000276:immunology; D000918:Antibody Specificity; D001483:Base Sequence; D015139:Blotting, Southern; D002135:Calcium-Binding Proteins / Q000096:biosynthesis* / Q000276:immunology / Q000378:metabolism; D003001:Cloning, Molecular; D004747:Entamoeba / Q000235:genetics; D004748:Entamoeba histolytica / Q000235:genetics* / Q000276:immunology; D004926:Escherichia coli / Q000378:metabolism*; D017125:Genes, Protozoan; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D015800:Protozoan Proteins / Q000096:biosynthesis* / Q000276:immunology / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000276:immunology; D013045:Species Specificity
D000913:Antibodies, Protozoan; D002135:Calcium-Binding Proteins; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; C075554:calcium-binding protein, Entamoeba histolytica