title: Expression, purification and characterisation of two variant cysteine peptidases from Trypanosoma congolense with active site substitutions.
abstract: Congopain, the major cysteine peptidase of Trypanosoma congolense is an attractive candidate for an anti-disease vaccine and target for the design of specific inhibitors. A complicating factor for the inclusion of congopain in a vaccine is that multiple variants of congopain are present in the genome of the parasite. In order to determine whether the variant congopain-like genes code for peptidases with enzymatic activities different to those of congopain, two variants were cloned and expressed. Two truncated catalytic domain variants were recombinantly expressed in Pichia pastoris. The two expressed catalytic domain variants differed slightly from one another in substrate preferences and also from that of C2 (the recombinant truncated form of congopain). Surprisingly, a variant with the catalytic triad Ser(25), His(159) and Asn(175) was shown to be active against classical cysteine peptidase substrates and inhibited by E-64, a class-specific cysteine protease inhibitor. Both catalytic domain clones and C2 had pH optima of either 6.0 or 6.5 implying that these congopain-like proteases are likely to be expressed and active in the bloodstream of the host animal.
authors.affiliation: School of Biochemistry, Genetics and Microbiology, University of KwaZulu-Natal, Scottsville, South Africa.
authors.forename: Davita
authors.initials: D
authors.lastname: Pillay
authors.forename: Alain F
authors.initials: AF
authors.lastname: Boulangé
authors.forename: Theresa H T
authors.initials: TH
authors.lastname: Coetzer
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D020134:Catalytic Domain; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics; D016415:Sequence Alignment; D014348:Trypanosoma congolense / Q000201:enzymology*
chemical_list: D003546:Cysteine Endopeptidases