Expression cloning in Escherichia coli and preparative isolation of the reductase coacting with chalcone synthase during the key step in the biosynthesis of soybean phytoalexins.
The cDNA for the reductase involved in the biosynthesis of 6'-deoxychalcone (4,2',4'-trihydroxychalcone), the first specific intermediate in the pathway to soybean phytoalexins, was cloned into the expression vector pKK233-2 and transformed into Escherichia coli. Using this source, about 5 mg of homogeneous reductase was isolated from 45 g of cells. The protein purification protocol differs completely from the scheme applied to soybean cell cultures. Size, N-terminal and specific enzyme activities were identical for the plant and E. coli protein. The pure protein is fairly stable, retaining 70% of initial activity after storage at 5 degrees C during 4 weeks. This protein is used for crystallization and in the study of its protein-protein interaction with chalcone synthase.
Welle
R
R
Lehrstuhl für Biochemie der Pflanzen, Biologisches Institut II, Universität Freiburg, Germany.
Schröder
J
J
D000217:Acyltransferases / Q000737:chemistry / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics*; D017343:Genes, Plant; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D010936:Plant Extracts / Q000096:biosynthesis* / Q000235:genetics; D012717:Sesquiterpenes; D013025:Glycine max / Q000737:chemistry / Q000201:enzymology / Q000235:genetics*; D013729:Terpenes; D000094162:Phytoalexins
D010936:Plant Extracts; D012717:Sesquiterpenes; D013729:Terpenes; D010088:Oxidoreductases; D000217:Acyltransferases; C020141:flavanone synthetase; D000094162:Phytoalexins