Synthesis, cloning, and expression in Escherichia coli of a spinach acyl carrier protein-I gene.
A synthetic gene of 268 bp encoding the 82 amino acid spinach acyl carrier protein (ACP)-I was constructed based on the known amino acid sequence. Two gene fragments, one encoding the amino-terminal portion and the other the carboxy-terminal portion of the protein, were assembled from synthetic oligonucleotides and inserted into the phage M13mp19. These partial gene constructions were joined and inserted into the plasmid pTZ19R. DNA sequencing confirmed the accuracy of the constructions. The synthetic gene was then subcloned into the Escherichia coli expression vector pKK233-2, under the control of the trc promoter. Western blot analysis and radioimmunoassay indicated that E. coli cells carrying this plasmid produced up to 6 mg/liter of a protein which was immunologically cross-reactive and similar in electrophoretic mobility to authentic spinach acyl carrier protein. The bacterial cells were able to attach the phosphopantetheine prosthetic group to the synthetic plant gene product allowing it to be acylated in vitro by acyl-ACP synthetase.
Beremand
P D
PD
Hannapel
D J
DJ
Guerra
D J
DJ
Kuhn
D N
DN
Ohlrogge
J B
JB
D000213:Acyl Carrier Protein / Q000096:biosynthesis / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D002848:Chromatography, DEAE-Cellulose; D003001:Cloning, Molecular; D003062:Codon; D004926:Escherichia coli / Q000235:genetics; D005796:Genes; D010446:Peptide Fragments / Q000235:genetics; D010944:Plants / Q000235:genetics*; D014170:Transformation, Genetic
D000213:Acyl Carrier Protein; D003062:Codon; D010446:Peptide Fragments