Toxoplasma gondii is a polyxenous parasite of the phylum Apicomplexa and the
etiological agent of the coccidian disease Toxoplasmosis. Toxoplasma gondii is
characterized by its unique gliding motility as well as its ability to infect virtually all
nucleated cells. The micronemes are a group of secretory organelles that contribute to
the ability of the parasite to recognize the surface of the host-cell prior to parasite
invasion via the secretion of adhesive protein complexes.
Previous attempts at obtaining structural data for several microneme proteins from
Toxoplasma gondii such as microneme protein TgMIC 2, 3 and 4 have so far been
unsuccessful due to the inability to produce soluble protein samples using the
prokaryotic expression system, Escherichia coli. This work is focussed upon the
adaptation of a eukaryotic expression system, Pichia pastoris, to producing
Apicomplexan microneme proteins which can cause problems in prokaryotic
expression systems due to its eukaryotic origin as well as its high cysteine content.
Modifications to the fermenter medium and standard screening protocols allowed for
the expression of microneme protein domain constructs in Pichia pastoris with yields
ranging from 0.1 to 1.2 mg/L in fermenter culture. Soluble protein samples were
produced for several constructs expressed in Pichia pastoris including full length
samples of TgMIC3 and TgMIC4 as well as the TSR56 pair in TgMIC2. In addition,
isotopically enriched soluble and folded protein samples were successfully produced
with a yield of 0.02 to 0.12 mg/L and were successfully subjected to analysis via 2D
NMR. Full length TgMIC4 was also successfully crystallized and resulted in crystals
which diffracted to 8.0Å.
Pichia pastoris has been successful in producing soluble microneme protein samples
and has the potential to alleviate the long-standing bottleneck in our efforts to
understand the mechanisms involved in Toxoplasma gondii host-cell invasion.