RENATURATION OF 1-AMINOCYCLOPROPANE-1-CARBOXYLATE SYNTHASE EXPRESSED IN ESCHERICHIA-COLI IN THE FORM OF INCLUSION-BODIES INTO A DIMERIC ANDCATALYTICALLY ACTIVE ENZYME

1-Aminocyclopropane-1-carboxylate (ACC) synthase is a key enzyme regul ating the biosynthesis of the plant hormone ethylene. A wound-inducibl e zucchini ACC synthase cDNA was isolated by reverse-transcription pol ymerase chain reaction (RT-PCR) and expressed in a heterologous Escher ichia coli BL21(DE3)-pLysS:pET30a protein expression system. A method was developed and optimized for the renaturation of the ACC synthase e xpressed in the form of inclusion bodies. The optimum conditions were found to be unfolding in a buffer containing 100 mM Mops, pH 9.5, 6 M urea, and 50 mM DTT, for 3 h at 4 degrees C and refolding by a combine d process of dialysis and dilution in 100 mM Mops, pH 8, 30 mM Chaps, and 5 mM GSH at a protein concentration of 45 mu g/ml. The purified en zyme has a specific activity of 90,000 U mg(-1) and exhibits an appare nt homogeneity on SDS-PAGE fractionation. Biochemical characterization of the refolded enzyme revealed a high degree of similarity to the en zyme purified from the soluble source. The refolded enzyme was found t o be a dimer with a native size of 110 kDa, a K-m of 23 mu M, and a V- max of 112,000 U mg(-1). (C) 1998 Academic Press.