Expression and deletion analysis of the Trypanosoma brucei rhodesiense cysteine protease in Escherichia coli.
Trypanosoma brucei, the cause of African sleeping sickness, differentiates in the mammalian bloodstream from a long, slender trypanosome into a short, stumpy trypanosome. This event is necessary for infection of the tsetse fly and maintenance of the life cycle. We have previously shown that the stumpy form contains 10- to 15-fold-greater cysteine protease activity than either the slender form or the insect midgut procyclic, and we have isolated a cDNA encoding the protease. In order to determine whether the cDNA encodes the developmentally regulated cysteine protease, we have purified the protease from trypanosomes and have made a polyclonal antiserum against it. The trypanosomal protease gene was then expressed in Escherichia coli with three different methionines within the pre- and propeptides acting as initiation sites. In each case, a protein was synthesized that was recognized by an antiserum specific for the developmentally regulated trypanosomal cysteine protease. The protein synthesized from the more upstream initiation site within the propeptide was proteolytically active. The recombinant protease and the trypanosomal enzyme were identical with respect to peptide substrates and protease inhibitors. The protein remained active when synthesized in a truncated form lacking the nine consecutive prolines and carboxy-terminus extension, indicating that the terminal 108 amino acids are not necessary for proteolytic activity.
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Department of Molecular Biology, Scripps Clinic and Research Foundation, La Jolla, California 92038.
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D000595:Amino Acid Sequence; D000818:Animals; D000913:Antibodies, Protozoan / Q000276:immunology; D001483:Base Sequence; D015153:Blotting, Western; D002872:Chromosome Deletion; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D008969:Molecular Sequence Data; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011817:Rabbits; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism; D013379:Substrate Specificity; D014346:Trypanosoma brucei brucei / Q000201:enzymology* / Q000235:genetics
D000913:Antibodies, Protozoan; D011994:Recombinant Proteins; D003546:Cysteine Endopeptidases