Expression and Automated Purification of Acetoacetyl CoA Thiolase from Sunflower Cotyledon

Abstract

In the sunflower (Helianthus annuus L.) cotyledons, two distinguishable thiolase activities have been identified: acetoacetyl CoA thiolase (AACT), specifically active during oxidation of short chain acetoacetyl CoA, and 3-oxoacyl CoA thiolase (OACT), active towards short, medium, and long-chain acyl CoAs. The purpose of this research was to optimize the purification of the AACT expressed in bacteria. Escherischia coli (E. coli) with the full-length sunflower AACT cDNA cloned into the expression vector pBAD-His B was induced for production of the AACT using 0.2% arabinose. Optimizing the conditions for protein purification is often an extremely empirical process requiring several iterations of a basic protocol each with specific changes. This iterative process was simplified using the programmable Äkta chromatography system from Amersham Pharmacia (now GE Healthcare). Programming this system for automatic sample injection and variation of elution conditions allows for optimization of the protein purification protocol to be achieved overnight as opposed to taking a day for each iteration done manually. Once optimized, soluble AACT was purified from E. coli bacteria in one step using a His HiTrap HP column containing Ni-Sepharose resin in conjunction with the Äkta chromatography system. AACT activity was detected in the induced bacteria, whereas no activity was observed with the uninduced control.