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.
Liu
Dong
D
Institute of Immunology, The Third Military Medical University, 30 Gaotanyan Street, Shapingba District, Chongqing 400038, People's Republic of China.
Zou
Liyun
L
Li
Wanling
W
Wang
Li
L
Wu
Yuzhang
Y
D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D004926:Escherichia coli / Q000235:genetics*; D015951:Factor Xa / Q000378:metabolism; D058619:Maltose-Binding Proteins; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D054334:Viral Regulatory and Accessory Proteins
D002352:Carrier Proteins; D058619:Maltose-Binding Proteins; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; D054334:Viral Regulatory and Accessory Proteins; C057628:hepatitis B virus X protein; D015951:Factor Xa