Purification and Biochemical Comparison of 1-Aminocyclopropane-1-carboxylic Acid Deaminase Proteins Expressed in Delayed Ripening Tomato and Escherichia coli:  Studies for a Food Safety Assessment

Tomato plants with delayed fruit ripening have been developed by introduction of the gene encoding the 1-aminocyclopropane-1-carboxylic acid deaminase (ACCd) protein into the tomato genome. The ACCd enzyme delays fruit ripening by decreasing ethylene synthesis. To facilitate safety assessments of the ACCd protein, the identical coding sequence used for tomato transformation was introduced into Escherichia coli to produce gram quantities of the protein. A method that included hydrophobic interaction and anion exchange chromatography was developed to purify approximately 2.5 g of ACCd. The E. coli-produced ACCd protein was demonstrated to be chemically and functionally equivalent to the ACCd protein expressed in and purified from delayed ripening tomato fruit. The proteins from each source had comparable apparent molecular weights, immunoreactivities, primary amino acid sequences, and enzymatic activities, and both proteins lacked glycosylation. These data validate the use of E. coli-produced ACCd protein to assess the safety of ACCd produced in delayed ripening tomatoes. Keywords: Delayed ripening tomato; 1-aminocyclopropane-1-carboxylic acid deaminase; protein purification; protein equivalence