Production in Escherichia coli of active Sorghum phosphoenolpyruvate carboxylase which can be phosphorylated.
Phosphoeolpyruvate carboxylase (PEPC)-deficient mutants of Escherichia coli have been complemented with a plasmid bearing a full-length cDNA encoding the C4-type form of Sorghum leaf PEPC. Transformed cells grew on minimal medium. Two clones were selected which produce a functional and full-sized enzyme protein as determined by activity assays, immunochemical behavior and SDS-PAGE. In addition, regulatory phosphorylation of immunopurified recombinant PEPC was observed when the enzyme was incubated with a partially purified plant PEPC kinase. These results establish that E. coli cells produce a genuine, phosphate-free, higher-plant PEPC. Application of immunoadsorbtion chromatography to bacterial extracts makes it possible to prepare highly pure protein available for biochemical studies.
Crétin
C
C
Laboratoire de Physiologie Végétale Moléculaire, URA CNRS D 1128, Université de Paris-Sud, Orsay, France.
Bakrim
N
N
Kéryer
E
E
Santi
S
S
Lepiniec
L
L
Vidal
J
J
Gadal
P
P
D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D010730:Phosphoenolpyruvate Carboxylase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D010766:Phosphorylation; D010957:Plasmids; D006109:Poaceae / Q000201:enzymology* / Q000235:genetics; D014170:Transformation, Genetic
D010730:Phosphoenolpyruvate Carboxylase