High-level expression and large-scale preparation of soluble HBx antigen from Escherichia coli.

The HBx (hepatitis B virus X protein) is a multifunctional regulator of cellular signal transduction and transcription pathways in host-infected cells. Evidence suggests that HBx has a critical role in the pathogenesis of hepatocellular carcinoma. However, the lack of efficient large-scale preparation methods for soluble HBx has hindered studies on the structure and function of HBx. Here, a new pMAL-c2x protein fusion and purification system was used for high-level expression of soluble HBx fusion protein. The high-purity fusion protein was obtained via amylose resin chromatography and Q-Sepharose chromatography. The untagged HBx was efficiently and rapidly purified by Sephadex G-75 chromatography after cleavage by Factor Xa at 23 degrees C. The purity of active HBx protein was >99% with a very stable secondary structure dominated by alpha-helix, beta-sheet and random structure. The purified HBx protein can be analysed to determine its crystal structure and function and its capabilities as an effective immunogen.