Heterologous expression, purification and characterisation of the extracellular domain of trypanosome invariant surface glycoprotein ISG75.
The invariant surface glycoprotein ISG75 is a transmembrane glycoprotein occurring on the surface of the bloodstream-form Trypanozoon. This study describes the expression and purification of the N-terminal extracellular domain of ISG75, a novel target for development of diagnostic tests for trypanosomosis. To facilitate disulfide formation in the cytoplasm, a 1287-bp cDNA fragment encoding ISG75 from Trypanosoma brucei gambiense was expressed in a thioredoxin reductase, glutathione oxidoreductase double mutant Escherichia coli strain. An accessory plasmid pRIL, providing the argI, ileY, and leuW tRNAs, was necessary for efficient heterologous translation of the ISG75 mRNA. The recombinant double-tagged (streptavidine and histidine) ISG75 was purified by two-step affinity chromatography. Addition of L-glutamic acid and L-arginine in the buffer solutions was crucial to stabilise the protein during purification. The purified soluble protein was characterised by circular dichroism spectroscopy, reverse-phase high pressure liquid chromatography and mass spectrometry. It has an alpha-helical folded conformation, is homogeneous and pure (99%). Furthermore, sera of Trypanosoma brucei-infected animals specifically recognise this recombinant ISG75; and rabbit antiserum raised against the recombinant ISG75 detects all species of the Trypanozoon subgenus in parasite preparations.
Department of Molecular and Cellular Interactions, Vrije Universiteit Brussel, Pleinlaan 2, Brussels, Belgium. thao.tran@vub.ac.be
Thao
T
Tran
Philippe
P
Büscher
Guy
G
Vandenbussche
Lode
L
Wyns
Joris
J
Messens
Henri
H
De Greve
D000595:Amino Acid Sequence; D000818:Animals; D002846:Chromatography, Affinity; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D006041:Goats; D013058:Mass Spectrometry; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010455:Peptides / Q000737:chemistry; D017434:Protein Structure, Tertiary; D015800:Protozoan Proteins / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D014346:Trypanosoma brucei brucei / Q000378:metabolism*
D008562:Membrane Glycoproteins; D010455:Peptides; D015800:Protozoan Proteins; C074691:ISG75 protein, Trypanosoma brucei