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Title: Structural studies on microneme proteins from Toxoplasma gondii
Author: Liew, Lloyd
ISNI:       0000 0004 2741 887X
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2013
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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.
Supervisor: Matthews, Steve Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral