Expression of the chalcone synthase gene from grape and preparation of an anti-CHS antibody

Flavonoids are closely related to a plant's antioxidative ability. Because chalcone synthase (CHS) is the first enzyme to act as part of the flavonoid biosynthesis pathway, its expression and regulation are important. Here we present the expression of a full-length chs cDNA with 1225 bp from grape seedlings as well as the preparation of an antibody against the expressed protein. A full-length chs cDNA was introduced into an expressed plasmid pET-30a(+) vector at the EcoRI and SalI restriction sites. pET-chs was found to be highly expressed in Escherichia coli BL21(DE3) pLysS cells with isopropyl-p-D-thiogalactoside (IPTG) induction. A fusion protein with the His center dot tag label was purified by Ni-NTA His center dot Bind Resin and then used as the antigen to immunize a New Zealand rabbit. The resulting antiserum was then further precipitated by 50% saturated ammonium sulfate and DEAE-Sepharose FF column chromatography to obtain the immunoglobulin G (IgG) fraction. The resulting antibody was found capable of immuno-recognizing the CHS of the crude protein extracts from different grape tissues with a molecular mass of 43 kDa. (c) 2006 Elsevier Inc. All rights reserved.