[Frontiers in Bioscience E2, 1134-1142, June 1, 2010]

Xiujun Zhang 1, Xi Ma2, Yongchang Xue3, Lijun Meng4, Baoling He1, Shengmei He1,3, Jie Zhao1, Yang Wang1, Wenping Yang1

1Department of Life Science, North China Coal medical college, No. 57 JianShe South Road, Tangshan 063000, China, 2State Key Laboratory of Animal Nutrition, China Agricultural University, No 2, Yuanmingyuan West Road, Beijing 100193, China, 3School of Biology and Food Technology, Dalian Polytechnic University, Dalian 116034, China, 1Department of Environment and Chemical Engineering, Tangshan College, Tangshan 063000, China

TABLE OF CONTENTS

1. Abstract
2. Introduction
3. Materials and Methods
3.1. Materials
3.2. cDNA cloning and vector construction
3.3. Expression of GST-AC3-33 fusion protein
3.4. Affinity purification of GST-AC3-33 fusion protein
3.5. Cleavage of GST-AC3-33 fusion protein
3.6. SDS-PAGE Electrophoresis
3.7. Polyclonal anti-AC3-33 antibody preparation
3.8. Western blot analysis
3.9. Cell culture and transient transfection with plasmids
3.10. Expression of mammalian recombinant protein
3.11. Dual-luciferase reporter assay
4. Results
4.1. Construction and identification of prokaryotic expression vector pGEX-4T-1-AC3-33
4.2. Optimization of expression condition of GST-AC3-33 fusion protein in E. coli BL21
4.3. Affinity purification of GST-AC3-33 fusion protein
4.4. Cleavage of GST-AC3-33 fusion protein and purification of recombinant AC3-33
4.5. Prokaryotic over-expression of AC3-33 suppresses AP-1 activity
5. Discussion
6. Acknowledgment
7. Reference

1. 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.