title: 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.
abstract: 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.
authors.affiliation: Lehrstuhl für Biochemie der Pflanzen, Biologisches Institut II, Universität Freiburg, Germany.
authors.forename: R
authors.initials: R
authors.lastname: Welle
authors.forename: J
authors.initials: J
authors.lastname: Schröder
mesh_terms: 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
chemical_list: D010936:Plant Extracts; D012717:Sesquiterpenes; D013729:Terpenes; D010088:Oxidoreductases; D000217:Acyltransferases; C020141:flavanone synthetase; D000094162:Phytoalexins