Expression and Purification of Sunflower Cotyledon Acetoacetyl CoA Thiolase Mutants

Abstract

Sunflower (Helianthus annuus L) cotyledons have two thiolase activities that have been identified previously. Thiolase II (acetoacetyl CoA thiolase) has specificity for the four‐carbon acetoacetyl CoA, resulting in the production of two Acetyl CoA molecules. Thiolase I (oxoacyl CoA thiolase) is active towards short, medium and long chain acyl CoAs. The purpose of this research was to optimize the expression and purification of mutant forms of Thiolase II from an E. coli bacterial expression system. Mutants generated by site‐directed mutagenesis were cloned into the pET151 expression vector, and this construct was transformed into BL‐21 E. coli that were induced for production of the Thiolase II with IPTG (Isopropyl β‐D‐1‐thiogalactopyranoside). The soluble Thiolase II was purified to apparent homogeneity using Ni‐NTA agarose affinity chromatography as indicated by a single band appearing on a Coomassie Blue stained SDS‐PAGE in the fraction eluted from the column with 100 mM imidazole.