Prokaryotic expression and characterization of human AC3-33 protein.
The transcription factor, AP-1, plays an important role in cellular proliferation, transformation and death. We previously showed that AC3-33 (GenBank name: c3orf33, FLJ31139), significantly inhibited transcriptional activity of AP-1. In this study, we report a method to express and purify AC3-33 in E. coli using glutathione-S-transferase (GST) fusion system. A GST-fusion protein was created by insertion of AC3-33 gene into a pGEX-4T-1 vector. The fusion protein, GST-AC3-33, was expressed in BL21 strain, and purified by GSH-affinity chromatography followed by thrombin cleavage. The digested product was further purified in a GSH-affinity column. After cleavage and purification, the recombinant AC3-33 protein exhibited the expected size of 29 kDa by SDS-PAGE and Western blotting and inhibited transcriptional activity of AP-1 in a dual-luciferase reporter assay. The bioactive recombinant GST-AC3-33, can be used to decipher the physiological and biochemical role of this protein.
Department of Life Science, North China Coal medical college, No 57 JianShe South Road, Tangshan 063000, China.
Xiujun
X
Zhang
Xi
X
Ma
Yongchang
Y
Xue
Lijun
L
Meng
Baoling
B
He
Shengmei
S
He
Jie
J
Zhao
Yang
Y
Wang
Wenping
W
Yang
D015153:Blotting, Western; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008565:Membrane Proteins; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
C552402:C3orf33 protein, human; D018076:DNA, Complementary; D008565:Membrane Proteins; D011506:Proteins; D011994:Recombinant Proteins