Maize recombinant C4-pyruvate,orthophosphate dikinase: Expression in Escherichia coli, partial purification, and characterization of the phosphorylatable protein |
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Authors: | Chris J Chastain Brent J Thompson Raymond Chollet |
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Institution: | (1) Department of Biology, Moorhead State University, 56563 Moorhead, MN, USA;(2) Department of Biochemistry, University of Nebraska-Lincoln, G.W. Beadle Center, 68588-0664 Lincoln, NE, USA |
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Abstract: | The gene for C4-pyruvate,orthophosphate dikinase (PPDK) from maize (Zea mays) was cloned into an Escherichia coli expression vector and recombinant PPDK produced in E. coli cells. Recombinant enzyme was found to be expressed in high amounts (5.3 U purified enzyme-activity liter-1 of induced cells) as a predominantly soluble and active protein. Biochemical analysis of partially purified recombinant PPDK showed this enzyme to be equivalent to enzyme extracted from illuminated maize leaves with respect to (i) molecular mass, (ii) specific activity, (iii) substrate requirements, and (iv) phosphorylation/inactivation by its bifunctional regulatory protein.Abbreviations DTT-
dithiothreitol
- FPLC-
fast-protein liquid chromatography
- HAP-
hydroxyapatite
- IPTG-
isopropyl- -thiogalactoside
- MOPS-
3-(N-morpholino)propanesulfonic acid
- PCR-
polymerase chain reaction
- PEP-
phosphoenolpyruvate
- PMSF-
phenylmethylsufonyl fluoride
- PPDK-
pyruvate,orthophosphate dikinase
- RP-
regulatory protein |
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Keywords: | C4 photosynthesis C4 plant maize (Zea mays) PPDK pyruvate orthophosphate dikinase recombinant enzyme |
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