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1: Protein Expr Purif. 1996 Sep;8(2):183-90.Click here to read Links

Cloning of chalcone-flavanone isomerase cDNA from Pueraria lobata and its overexpression in Escherichia coli.

Terai Y, Fujii I, Byun SH, Nakajima O, Hakamatsuka T, Ebizuka Y, Sankawa U.

Faculty of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113, Japan.

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.

PMID: 8812858 [PubMed - indexed for MEDLINE]