Pentatricopeptide repeat (PPR) proteins are eukaryotic RNA binding proteins that are
especially prevalent in terrestrial plants. Members of the PPR family in plants can be
classified into two distinct subfamilies, the P subfamily and the PLS subfamily. A subclass of
PLS subfamily PPR proteins contain a novel C-terminal domain, named the DYW deaminase
domain due to its conserved terminal aspartate (D), tyrosine (Y), tryptophan (W) triplet.
DYW type PPR proteins are implicated as trans-factors in C to U RNA editing in plant
chloroplasts and mitochondria, although structural and mechanistic information about this
process is lacking. To address this, we have expressed and purified a recombinant
Arabidopsis thaliana DYW-type protein, AEF3, as an N-terminal His 6 -Maltose Binding
Protein (MBP) fusion construct on amylose resin. However, Size Exclusion Chromatography
coupled Multi-Angle Light Scattering (SEC-MALS) data suggests that this fusion protein
aggregates into large complexes in the MDa mass range (> 10 6 Da). In addition, the fusion
protein precipitates upon removal of the His 6 -MBP fusion tag with Tobacco Etch Virus
(TEV) protease when analysed by SDS-PAGE likely linked to its aggregating behaviour as a
purified fusion. This behaviour upon TEV protease treatment has been utilised to develop an
SDS-PAGE based assay to identify conditions where the aggregation state of the protein has
been improved. Stable cleaved AEF3 is desired to be used for X-ray Crystallography, and for
Small Angle X-ray Scattering experiments, to obtain structural data on this DYW-type PPR
protein and it’s RNA binding and editing mechanisms.