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 1225bp 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-beta-d-thiogalactoside (IPTG) induction. A fusion protein with the His.tag label was purified by Ni-NTA His. 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 43kDa.
Tian
Li
L
College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China.
Kong
Wei Fu
WF
Pan
Qiu Hong
QH
Zhan
Ji Cheng
JC
Wen
Peng Fei
PF
Chen
Jian Ye
JY
Wan
Si Bao
SB
Huang
Wei Dong
WD
D000217:Acyltransferases / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D000818:Animals; D000918:Antibody Specificity; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology; D007074:Immunoglobulin G / Q000276:immunology / Q000302:isolation & purification*; D010940:Plant Proteins / Q000276:immunology / Q000302:isolation & purification*; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D036226:Seedlings / Q000201:enzymology; D027843:Vitis / Q000201:enzymology*
D007074:Immunoglobulin G; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D000217:Acyltransferases; C020141:flavanone synthetase