High-level bacterial expression and purification of apicomplexan micronemal proteins for structural studies

Saouros, S., Blumenschein, T. M. A. ORCID: https://orcid.org/0000-0002-4932-5178, Sawmynaden, K., Marchant, J., Koutroukides, T., Liu, B., Simpson, P., Carpenter, E. P. and Matthews, S. J. (2007) High-level bacterial expression and purification of apicomplexan micronemal proteins for structural studies. Protein and Peptide Letters, 14 (5). pp. 411-415. ISSN 1875-5305

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Abstract

Micronemal protein secretion from apicomplexan parasites like Toxoplasma gondii is responsible for the directional attachment of the parasite to the host in a step critical in invasion. Despite the medical importance and the justification of micronemal proteins as drug targets, very little is known about their specific mechanisms of host attachment and the nature of their host receptors. Here we describe the cloning, high-level expression in Escherichia coli and purification of the N-terminal domain of the adhesive protein MIC1 (MIC1-NT) from Toxoplasma gondii as a thioredoxin fusion protein; a protein fragment with 16 cysteines and 8 potential disulphide bonds. The final, cleaved product is close to 100% pure by SDS-PAGE and the yield was about 15mg/L. The protein fragment is soluble, stable and suitable for structural and functional studies. MIC1-NT was characterised by mass spectroscopy and size-exclusion chromatography to prove its presence in monomeric form. The 1D 1H and 2D (1H-15N) HSQC spectra reveal that the protein is well structured. The same strategy was applied to two additional cysteine rich micronemal proteins giving similar results to MIC1-NT. Our results demonstrate that our approach can have a wider application in the recombinant expression, structural and functional studies of the extended family of micronemal proteins.

Item Type:	Article
Faculty \ School:	Faculty of Science > School of Chemistry (former - to 2024)
UEA Research Groups:	Faculty of Science > Research Groups > Chemistry of Life Processes Faculty of Science > Research Centres > Centre for Molecular and Structural Biochemistry Faculty of Science > Research Groups > Biophysical Chemistry (former - to 2017)
Depositing User:	Rachel Smith
Date Deposited:	15 Feb 2011 10:59
Last Modified:	24 Sep 2024 09:37
URI:	https://ueaeprints.uea.ac.uk/id/eprint/21383
DOI:	10.2174/092986607780782876

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