[Induced expression of Arabidopsis thaliana WUSCHEL in Escherichia coli, affinity protein purification and polyclonal antibody preparation].
We constructed a His-tagged prokaryotic expression vector of WUSCHEL gene of Arabidopsis thaliana, pET-31b(+)-WUS-His(6). The induction condition of the fusion protein expression in Escherichia coli was optimized. After purified by affinity chromatography, the recombinant WUS protein was resolved by renaturation of gradient urea dialysis, then used as antigen to immune rabbit to prepare polyclonal antibody. The rabbit anti-WUS antibody titer and specificity were analyzed and confirmed by agarose immunodiffusion testing; the antiserum sensitivity was assayed by dot blot and Western blotting. The results showed that the A. thaliana WUS prokaryotic expression vector was successfully constructed, and the optimized protein expression induction condition in E. coli was 0.5 mmol/L IPTG (isopropy-beta-D-thiogalactoside) at 28 degrees C for 10 hours. The purity of the affinity purified protein was higher than 96%, and the prepared polyclonal antibody was with high specificity and sensitivity, it was able to detect protein antigen at ng level.
Wang
Zeng
Z
College of Agriculture and Biotechnology, China Agricultural University, Beijing 100193, China.
Dai
Ru
R
Zhang
Jiangwei
J
Chen
Shangwu
S
Zhang
Wen
W
Ma
Huiqin
H
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000302:isolation & purification / Q000378:metabolism; D017360:Arabidopsis / Q000235:genetics*; D029681:Arabidopsis Proteins / Q000096:biosynthesis* / Q000235:genetics; D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics; D018398:Homeodomain Proteins / Q000096:biosynthesis* / Q000235:genetics; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000096:biosynthesis / Q000235:genetics; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*
D000906:Antibodies; D029681:Arabidopsis Proteins; D018398:Homeodomain Proteins; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; C116421:WUSCHEL protein, Arabidopsis