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The first purification of functional proteins from the unculturable, genome reduced, bottlenecked alpha-proteobacterium Candidatus Liberibacter solanacearum.
Phytopathology 2019 March 20
'Candidatus Liberibacter solanacearum' (CLso) is an unculturable α-proteobacterium that is the causal agent of zebra chip disease of potato-a major problem in potato-growing areas, as it affects growth and yield. Developing effective treatments for CLso has been hampered by the difficulty in functionally characterizing the proteins of this organism, largely because they are not easily expressed and purified in standard expression systems. CLso has a reduced genome and its proteins are predicted to be prone to instability and aggregation. Among intracellular-dwelling bacteria, chaperone proteins are conserved and overexpressed to buffer against problems in protein folding. We mimicked this approach for expressing and purifying CLso proteins in Escherichia coli, by co-expressing them with chaperones. Neither of the representative CLso enzymes, dihydrodipicolinate synthase (key in lysine biosynthesis) and pyruvate kinase (involved in glycolysis), were overexpressed in standard E. coli expression plasmids or strains. However, soluble dihydrodipicolinate synthase was successfully co-expressed with GroEL/GroES, whilst soluble pyruvate kinase was successfully co-expressed with either GroEL/GroES, dnaK/dnaJ/grpE, or a trigger factor. Both enzymes, believed to be key proteins for the organism, were purified by a combination of affinity chromatography and size exclusion chromatography. Additionally, both CLso enzymes are active and have the canonical tetrameric oligomeric structure in solution, consistent with other bacterial orthologues. This is the first study to successfully isolate and functionally characterize proteins from CLso. Thus, we provide a general strategy for characterizing its proteins, enabling new research and drug discovery programmes to study and manage the pathogenicity of the organism.
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