Modern drug discovery is a target-driven approach in which a particular protein such as an enzyme is implicated in the disease process. Commonly, small-molecule drugs are identified using screening, rational design, and structural biology approaches. Drug screening, testing and optimization is typically conducted in vitro, and copious amounts of protein are required. The advent of recombinant DNA technologies has resulted in a rise in proteins purified by affinity techniques, typically by incorporating an "affinity tag" at the N- or C-terminus. Use of these tagged proteins and affinity techniques comes with a host of issues. This chapter describes the production of an untagged enzyme, α-methylacyl-CoA racemase (MCR) from Mycobacterium tuberculosis, using a recombinant E. coli system. Purification of the enzyme on a 100 mg scale using tandem anion-exchange chromatographies (DEAE-sepharose and RESOURCE-Q columns), and size-exclusion chromatographies is described. A modified protocol allowing the purification of cationic proteins is also described, based on tandem cation-exchange chromatographies (using CM-sepharose and RESOURCE-S columns) and size-exclusion chromatographies. The resulting MCR protein is suitable for biochemical and structural biology applications. The described protocols have wide applicability to the purification of other recombinant proteins and enzymes without using affinity chromatography.
Keywords: Anion-exchange chromatography; Cation-exchange chromatography; Expression and purification of recombinant proteins; Recombinant E. coli; SDS-PAGE; Size-exclusion chromatography; α-Methylacyl-CoA racemase.
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