Cloning of chalcone-flavanone isomerase cDNA from Pueraria lobata and its overexpression in Escherichia coli.
Chalcone-flavanone isomerase (CHI) cDNA was isolated from Pueraria lobata by combination of cDNA library screening using Phaseolus vulgaris CHI cDNA as a probe and polymerase chain reaction techniques. Analysis of nucleotide sequence of the cloned cDNA revealed a 675-bp open reading frame encoding a 225-amino-acid polypeptide with a molecular weight of 23,803 Da. The CHI cDNA coding region was cloned into pET-3d expression vector and successfully overexpressed in Escherichia coli cells as active CHI enzyme. The recombinant CHI was then purified to apparent homogeneity by DEAE-cellulose column chromatography. Replacement of Cys-119 with Ala was carried out by site-directed mutagenesis and the result that the mutant CHI showed CHI enzyme activity confirmed that Cys-119 is not involved in the CHI catalytic active site.
Terai
Y
Y
Faculty of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113, Japan.
Fujii
I
I
Byun
S H
SH
Nakajima
O
O
Hakamatsuka
T
T
Ebizuka
Y
Y
Sankawa
U
U
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002848:Chromatography, DEAE-Cellulose; D003001:Cloning, Molecular; D016384:Consensus Sequence / Q000235:genetics; D017124:Conserved Sequence / Q000235:genetics; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D007887:Fabaceae / Q000737:chemistry / Q000235:genetics; D005419:Flavonoids / Q000096:biosynthesis / Q000378:metabolism; D015870:Gene Expression / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D019753:Intramolecular Lyases; D007535:Isomerases / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D010944:Plants / Q000201:enzymology; D010946:Plants, Medicinal; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D017421:Sequence Analysis; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D017931:DNA Primers; D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D005419:Flavonoids; D011994:Recombinant Proteins; D013439:Sulfhydryl Reagents; D007535:Isomerases; D019753:Intramolecular Lyases; C019865:chalcone isomerase