Expression and Purification of Sunflower Thiolase Mutants

Abstract

Sunflower (Helianthus annus 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 these two thiolases from an E. coli bacterial expression system. Mutants generated by site‐directed mutagenesis were cloned into the pET151 expression vector, and constructs were transformed into BL‐21 E. coli that were induced for production of the thiolases with IPTG (Isopropyl β‐D‐1‐thiogalactopyranoside). The soluble thiolases were purified and were used to characterize their enzymatic properties.