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Cloning of Chalcone–Flavanone Isomerase cDNA fromPueraria lobataand Its Overexpression inEscherichia coli

https://doi.org/10.1006/prep.1996.0091Get rights and content

Abstract

Chalcone–flavanone isomerase (CHI) cDNA was isolated fromPueraria lobataby combination of cDNA library screening usingPhaseolus vulgarisCHI 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 inEscherichia colicells 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.

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  • Functional and genomic characterization of a wound- and methyl jasmonate-inducible chalcone isomerase in Eremochloa ophiuroides [Munro] Hack

    2019, Plant Physiology and Biochemistry
    Citation Excerpt :

    Type-I CHIs are found in non-leguminous plants and only isomerize 6′-hydroxychalcone to 5-hydroxyflavanone, whereas type-II CHIs are specialized in legume plants and accept both 6′-hydroxychalcone and 6′-deoxychalcone as substrates to produce 5-hydroxyflavanone and 5-deoxyflavanone, respectively (Fig. 1). The CHI activities of crude proteins and the functional characterizations of CHI complementary DNAs (cDNAs) have been documented in various plant species, including A. thaliana (Shirley et al., 1995), Glycine max (Bednar and Hadcock, 1988), Zea mays (Grotewold and Peterson, 1994), Ipomoea batatas (Guo et al., 2015), Saussurea medusa (Li et al., 2006), Oryza sativa (Shih et al., 2008), Trigonella foenum-graecum (Qin et al., 2011), Ginkgo biloba (Cheng et al., 2011), Citrus sinensis (Fouché and Dubery, 1994), Petunia hybrida (Van Tunen and Mol, 1987), and Pueraria lobata (Terai et al., 1996). Type-II CHIs are most likely present only in leguminous plants, some of which have shown the exceptional genomic feature of having both type-I and type-II CHIs (Kimura et al., 2001), or a gene cluster with four type-I and –II CHIs in Lotus japonicus (Shimada et al., 2003).

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1

Current address: Faculty of Pharmaceutical Sciences, Science University of Tokyo, Funagawara-cho 12, Shinjuku-ku, Tokyo 162, Japan.

2

To whom correspondence should be addressed. E-mail: yebiz@ mol.f.u-tokyo.ac.jp.

3

Current address: Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, Sugitani 2630, Toyama 930-01, Japan.

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