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.
Zhang
Xiujun
X
Department of Life Science, North China Coal medical college, No 57 JianShe South Road, Tangshan 063000, China.
Ma
Xi
X
Xue
Yongchang
Y
Meng
Lijun
L
He
Baoling
B
He
Shengmei
S
Zhao
Jie
J
Wang
Yang
Y
Yang
Wenping
W
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