Heterologous expression, purification and characterisation of the extracellular domain of trypanosome invariant surface glycoprotein ISG75

Thao Tran, P. Büscher, Guy Vandenbussche, Lode Wyns, Joris Messens, Henri De Greve

Onderzoeksoutput: Articlepeer review

27 Citaten (Scopus)

Samenvatting

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 gambiensewas 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 lglutamic
acid and l-arginine in the buffer solutionswas 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.
Originele taal-2English
Pagina's (van-tot)247-254
Aantal pagina's8
TijdschriftJournal of Biotechnology
Volume135
Nummer van het tijdschrift3
StatusPublished - 30 jun 2008

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