Renaturation of 1-aminocyclopropane-1-carboxylate synthase expressed in Escherichia coli in the form of inclusion bodies into a dimeric and catalytically active enzyme.
1-Aminocyclopropane-1-carboxylate (ACC) synthase is a key enzyme regulating the biosynthesis of the plant hormone ethylene. A wound-inducible zucchini ACC synthase cDNA was isolated by reverse-transcription polymerase chain reaction (RT-PCR) and expressed in a heterologous Escherichia coli BL21(DE3)pLysS:pET30a protein expression system. A method was developed and optimized for the renaturation of the ACC synthase expressed 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 combined process of dialysis and dilution in 100 mM Mops, pH 8, 30 mM Chaps, and 5 mM GSH at a protein concentration of 45 microg/ml. The purified enzyme has a specific activity of 90,000 U mg-1 and exhibits an apparent homogeneity on SDS-PAGE fractionation. Biochemical characterization of the refolded enzyme revealed a high degree of similarity to the enzyme purified from the soluble source. The refolded enzyme was found to be a dimer with a native size of 110 kDa, a Km of 23 microM, and a Vmax of 112,000 U mg-1.
Huxtable
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S
Department of Biology, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong.
Zhou
H
H
Wong
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Li
N
N
D001483:Base Sequence; D002021:Buffers; D019662:Cucurbitaceae / Q000201:enzymology* / Q000235:genetics; D017931:DNA Primers / Q000737:chemistry; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics; D018506:Gene Expression Regulation, Plant / Q000235:genetics*; D006863:Hydrogen-Ion Concentration; D002479:Inclusion Bodies / Q000201:enzymology* / Q000378:metabolism; D007700:Kinetics; D008190:Lyases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D009994:Osmolar Concentration; D016133:Polymerase Chain Reaction; D017510:Protein Folding; D013997:Time Factors
D002021:Buffers; D017931:DNA Primers; D008190:Lyases; C021773:1-aminocyclopropanecarboxylate synthase