Prokaryotic expression and characterization of human AC3-33 protein

Front Biosci (Elite Ed). 2010 Jun 1;2:1134-42. doi: 10.2741/e172.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Blotting, Western
  • Chromatography, Affinity
  • Cloning, Molecular
  • DNA, Complementary
  • Electrophoresis, Polyacrylamide Gel
  • Humans
  • Membrane Proteins
  • Proteins / genetics
  • Proteins / metabolism*
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism

Substances

  • C3orf33 protein, human
  • DNA, Complementary
  • Membrane Proteins
  • Proteins
  • Recombinant Proteins