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.
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Abbreviations
- DTT-:
-
dithiothreitol
- FPLC-:
-
fast-protein liquid chromatography
- HAP-:
-
hydroxyapatite
- IPTG-:
-
isopropyl-β-thiogalactoside
- MOPS-:
-
3-(N-morpholino)propanesulfonic acid
- PCR-:
-
polymerase chain reaction
- PEP-:
-
phosphoenolpyruvate
- PMSF-:
-
phenylmethylsufonyl fluoride
- PPDK-:
-
pyruvate,orthophosphate dikinase
- RP-:
-
regulatory protein
References
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Chastain, C.J., Thompson, B.J. & Chollet, R. Maize recombinant C4-pyruvate,orthophosphate dikinase: Expression in Escherichia coli, partial purification, and characterization of the phosphorylatable protein. Photosynth Res 49, 83–89 (1996). https://doi.org/10.1007/BF00029430
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DOI: https://doi.org/10.1007/BF00029430