ENHANCED HIGH-LEVEL EXPRESSION OF SOLUBLE 1-AMINOCYCLOPROPANE-1-CARBOXYLASE SYNTHASE AND RAPID PURIFICATION BY EXPANDED-BED ADSORPTION

1-Aminocyclopropane-1-carboxylate (ACC) synthase is a hey enzyme regul ating the biosynthesis of the plant hormone ethylene. Expression of AC C synthase in Escherichia coli can result in the production of a large proportion of the enzyme in the form of insoluble aggregates (inclusi on bodies). We investigated the effect on the soluble expression in E. coli of tomato and zucchini ACC synthases, by manipulation of the ind uction conditions, changing the vector, and deletions in the amino aci d sequence. Manipulation of the induction conditions did not influence the soluble expression; however, soluble expression increased signifi cantly when the enzyme was cloned into vector pET11d, in comparison to the other vector used, pET30a. It was also found that when ACC syntha se with a portion of the C-terminus deleted was inserted into pET11d, the soluble expression was further enhanced in comparison to that of t he full length. Structural and functional analysis of ACC synthase req uires the purification of milligram quantities of protein to homogenei ty. The development of a faster and simpler protocol for the purificat ion of ACC synthase is highly desirable due to the extreme lability of the enzyme. C-terminal truncated tomato ACC synthase was overexpresse d in E. coli pET11d and purified by expanded-bed adsorption and hydrox ylapatite FPLC. This improved two-step purification protocol allows fo r rapid, high-level purification with a significantly improved yield i n comparison to the multistage purification it replaces. 15.7 mg of hi ghly purified tomato ACC synthase del-l were obtained from 2 L of cell s in comparison to 2 mg hom 10 L using a multistage purification. This represents a 40-fold improvement in yield. Antibodies were raised aga inst C-terminal deleted ACC synthase. The antibodies were purified by epitope-specific affinity chromatography and used to assess the identi ty and purity of the C-terminal deleted tomato ACC synthase purified b y expanded-bed adsorption, (C) 1998 Academic Press.