Expression, purification, crystallization and X-ray diffraction studies of a novel root-induced secreted protein from Trichoderma virens

Small secreted cysteine-rich proteins (SSCPs) from fungi play an important role in fungi – host interactions. The plant-beneficial fungi Trichoderma spp. are in use worldwide as biocontrol agents and protect the host plant from soil-borne as well as foliar pathogens. Recently, a novel SSCP, Tsp1, has been identified in the secreted protein pool of T. virens and is overinduced upon its interaction with the roots of the maize plant. The protein was observed to be well conserved in the Ascomycota division of fungi, and its homologs are present in many plant-pathogenic fungi such as Fusarium oxysporum and Magnaporthe oryzae. However, none of these homologs have yet been characterized. Recombinant Tsp1 protein has been expressed and purified using an Escherichia coli expression system. The protein, with four conserved cysteines, forms a dimer in solution as observed by size-exclusion chromatography. The dimerization, however, does not involve disulfide bonds. Circular-dichroism data suggested that the protein has a β -strand-rich secondary structure that matched well with the secondary structure predicted using bioinformatics methods. The protein was crystallized using sodium malonate as a precipitant. The crystals diffracted X-rays to 1.7   Å resolution and belonged to the orthorhombic space group P212121 (Rmeas = 5.4%), with unit-cell parameters a = 46.3, b = 67.0, c = 173.2   Å . The Matthews coefficient (VM) of the crystal is 2.32   Å 3   Da − 1, which correspo...
Source: Acta Crystallographica Section F - Category: Biochemistry Authors: Tags: Trichoderma virens effector secreted cysteine-rich proteins protein crystallization research communications Source Type: research