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Cloning and sequencing of the cysteine protease cDNA from Trypanosoma brucei rhodesiense.

The Trypanosoma brucei rhodesiense (WRATat 1.1) cysteine protease cDNA was obtained from a cDNA library in GTl (provided by Dr. John Donelson, Univ. of Iowa) by screening with a 495 base pair DNA molecule encoding the active site of the T. brucei cysteine protease (1). The 1.7 kb insert was inserted into the EcoRl site of the Bluescript plasmid (Stratagene, La Jolla, CA) and designated pTCP. Deletions of the insert were made with Exonuclease HI and Mung Bean Nuclease as described by the manufacturer (Stratagene, La Jolla, CA). Sequencing of the insert by the dideoxy-nucleotide chain termination method was performed on the sense strand from the endpoint of the deletions. The other strand was sequenced by using 20-mer primers made with an Applied Biosystems DNA synthesizer. The cDNA encodes both a pre and pro peptide and the cysteine protease sequence. There is also an unusual carboxy-terminus extension that begins with 9 consecutive prolines and continues with a further 100 amino acids including 8 cysteines. The sequence we have obtained is nearly identical to that obtained for Trypanosoma brucei by Mottram et al. (2). On comparison we find 15 nucleotide substitutions resulting in only 6 amino acid substitutions, which are underlined in the amino acid sequence.