Abstract |
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.
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Authors | S Huxtable, H Zhou, S Wong, N Li |
Journal | Protein expression and purification
(Protein Expr Purif)
Vol. 12
Issue 3
Pg. 305-14
(Apr 1998)
ISSN: 1046-5928 [Print] United States |
PMID | 9535697
(Publication Type: Comparative Study, Journal Article, Research Support, Non-U.S. Gov't)
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Copyright | Copyright 1998 Academic Press. |
Chemical References |
- Buffers
- DNA Primers
- Lyases
- 1-aminocyclopropanecarboxylate synthase
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Topics |
- Base Sequence
- Buffers
- Cucurbitaceae
(enzymology, genetics)
- DNA Primers
(chemistry)
- Dimerization
- Escherichia coli
(genetics)
- Gene Expression Regulation, Plant
(genetics)
- Hydrogen-Ion Concentration
- Inclusion Bodies
(enzymology, metabolism)
- Kinetics
- Lyases
(chemistry, genetics, isolation & purification, metabolism)
- Osmolar Concentration
- Polymerase Chain Reaction
- Protein Folding
- Time Factors
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