title: Recombinant expression of trypanosome surface glycoproteins in Pichia pastoris for the diagnosis of Trypanosoma evansi infection.
abstract: Serodiagnosis of surra, which causes vast economic losses in livestock, is still based on native antigens purified from bloodstream form Trypanosoma (T.) evansi grown in rodents. To avoid the use of laboratory rodents in antigen preparation we expressed fragments of the invariant surface glycoprotein (ISG) 75, cloned from T. brucei gambiense cDNA, and the variant surface glycoprotein (VSG) RoTat 1.2, cloned from T. evansi gDNA, recombinantly in Pichia (P.) pastoris. The M5 strain of this yeast has an engineered N-glycosylation pathway resulting in homogenous Man5GlcNAc2 N-glycosylation which resembles the predominant Man9-5GlcNAc2 oligomannose structures in T. brucei. The secreted recombinant antigens were affinity purified with yields of up to 10mg and 20mg per liter cell culture of rISG 7529-465-E and rRoTat 1.223-385-H respectively. In ELISA, both recombinant proteins discriminated between pre-immune and immune serum samples of 25 goats experimentally infected with T. evansi. The diagnostic potential of rRoTat 1.223-385-H but not of rISG 7529-465-E was confirmed with sera of naturally infected and control dromedary camels. The results suggest that rRoTat 1.223-385-H expressed in P. pastoris requires further evaluation before it could replace native RoTat 1.2 VSG for serodiagnosis of surra, thus eliminating the use of laboratory animals for antigen production.
authors.affiliation: Department of Biomedical Sciences, Unit of Parasite Diagnostics, Institute of Tropical Medicine, Nationalestraat 155, B-2000 Antwerp, Belgium; Laboratory for Molecular Plant Physiology and Biotechnology, Department of Biology, University of Antwerp, Groenenborgerlaan 171, B-2020 Antwerp, Belgium. Electronic address: sroge@itg.be.
authors.forename: S
authors.initials: S
authors.lastname: Rogé
authors.forename: N
authors.initials: N
authors.lastname: Van Reet
authors.forename: S
authors.initials: S
authors.lastname: Odiwuor
authors.forename: T
authors.initials: T
authors.lastname: Tran
authors.forename: K
authors.initials: K
authors.lastname: Schildermans
authors.forename: S
authors.initials: S
authors.lastname: Vandamme
authors.forename: I
authors.initials: I
authors.lastname: Vandenberghe
authors.forename: W
authors.initials: W
authors.lastname: Vervecken
authors.forename: K
authors.initials: K
authors.lastname: Gillingwater
authors.forename: F
authors.initials: F
authors.lastname: Claes
authors.forename: B
authors.initials: B
authors.lastname: Devreese
authors.forename: Y
authors.initials: Y
authors.lastname: Guisez
authors.forename: P
authors.initials: P
authors.lastname: Büscher
mesh_terms: D000818:Animals; D004283:Dog Diseases / Q000517:prevention & control; D004285:Dogs; D005260:Female; D005786:Gene Expression Regulation / Q000502:physiology*; D010843:Pichia / Q000378:metabolism*; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors; D014345:Trypanosoma / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D015800:Protozoan Proteins
keywords: ISG 75; Pichia pastoris; Recombinant; RoTat 1.2 VSG; Surra; diagnosis