Expression And Purification Of Hepatitis B Virus X Protein And Establishment Of Models Of HBV-host Interactome

Chronic infection of hepatitis B virus (HBV) is one of the major causes of hepatocellular carcinoma (HCC) while HBV-related HCC is among the top 10 most common cancers in the world. At present, approximately 200 million people are chronically infected, so that HBV is still a major problem to human health. Although the pathogenesis of HBV-related HCC has not been fully described, evidence suggests that HBx plays a crucial role in the pathogenesis of HCC. HBx is a multifunctional regulator that modulates transcription, signal transduction, cell cycle progress, protein degradation pathways, apoptosis, and genetic stability by directly or indirectly interacting with host factors.The HBx protein consists of 154 amino acids and possesses a molecular weight of~17 kDa. The nine cysteines of HBx are important for maintaining its structure. Eight of the nine cysteines are disulphide linked in an interesting pattern. Each cysteine is linked to the fourth cysteine in a sequential manner and the last cysteine is free; the disulphide linkages are between Cys7 and Cys78, Cys17 and Cys115, Cys61 and Cys137, Cys69 and Cys143 while Cys148 is free. The structure makes it to become unplastic after renaturation. However, previous results showed that the whole HBx or truncated HBx were expressed in form of inclusion body in Escherichia coli. After denaturation and renaturation, the structure of HBx might be impacted partially, but it still have some functions binding with RNA and p53 protein. This suggests primary structure of HBx expressed in Escherichia coli is right and the strategy is feasible. When HBx was expressed in a baculovirus expression system, cell fractionation experiments revealed that only a minor part of HBx is detectable in a soluble form in the fraction, whereas most of the protein forms intracellular aggregates. Analysis of electrospray ionization mass spectrometry revealed that renaturational HBx only have three disulphides.Therefore, it is vital to express right and soluble HBx protein for structural and functional study of HBx.It is a key point for successfully right and soluble expression of HBx protein to choose suitable plasmid and host bacterium. Based on previous researches, we select pET32a plasmid with S tag and His tag,pQE30 plasmid with His tag and pMAL-c2x plasmid with MBP tag to express HBx protein in BL21(DE3),TB1 and JM109. The result showed that only the fusion protein with MBP tag is completely soluble in JM109. This demonstrates MBP tag is helpful for the solubility of HBx. We cultured 15L bacterium, collected precipitation and purify 60mg/ml fusion protein by sonication, centrifugalization, affinity column and ion exchange column. After the fusion protein was digested by factor Xa at 23℃in 48 hours, the solution become semiopaque. To overcome this problem, we dialyzed the fusion protein cleavage mixture vs. 20 mM Tris-HCl, 25 mM NaCl,1M Urea, 0.05% SDS, pH 10.0, then separated HBx by sephadex G-75 column. The concentration of HBx protein is approximately 0.5mg/ml. This serve as structural study of HBx protein in the future.HBV infection is one of the major causes of HCC in humans in the world. It spreads through humoral, sexual, and mather to child pathways. The human Hepatitis B Virus (HBV) is a double-stranded DNA virus that belongs to the family of hepadnaviruses and that causes acute and chronic viral hepatitis. An incomplete open circular DNA genome of HBV is consisted of a full length 3.2-kb minus strand and an incomplete plus strand. The viral genome is organized into 4 overlapping transcription units controlled by 4 independent promoters, yielding 4 extensively overlapping viral RNAs(3.5, 2.4, 2.1, and 0.7kb in length), and encoding seven proteins in the cytoplasm that are P, S, C, preS1, preS2, preC, and X proteins. The 3.5kb transcript produces the polymerase, core, and precore proteins and serves as the pregenomic RNA template that is reverse transcribed as the first step in viral genome replication. The 2.4- and 2.1-kb transcripts produce the large, middle, and small envelope proteins(S, preS1 and preS2). The 3 HBV envelope proteins share common carboxy termini and display progressive amino terminal extensions. The 0.7-kb transcript produces the X protein, which has transcriptional transactivating potential.HBV constantly adapts and modulates host environment by virus-host interactions during infection, replication and proliferation. Data for virus-host interactions allow the thorough characterization of the life cycles of viruses and have the potential to reveal important interactions that could be targeted for drug therapy. For this purpose, a TAP tagging approach was used to assess stable host cell interactions formed by HBV proteins when expressed in human HepG2 cells. Firstly, we constructed the pCR-XL-TOPO-HBV plasmid including the whole genome of HBV , then obtained P, S, preS1, preS2, C, preC, and X genes by PCR using this plasmid as template. Secondly, we constructed the pBT-TAP-IRES-BIRA vector with biotin tag at N-termini and TAP tag at C-termini as the control. Finally, we inserted the seven genes into the pBT-TAP-IRES-BIRA vector at SnaB I site respectively and constructed pBTIB-P, pBTIB-S,pBTIB-C, pBTIB-X, pBTIB-preC, pBTIB-preS1, and pBTIB-preS2 vectors. To reduce the effect on targeting proteins with the tags, we added GSGGSG in two sides of SnaB I site.The pBT-TAP-IRES-BIRA, pBTIB-X, pBTIB-P, pBTIB-S, pBTIB-C, pBTIB-preC, pBTIB-preS1, and pBTIB-preS2 vectors were transformed into PT67 cells by screening with 3.5μg/ml puromycin and eight kinds of viruses were packaged and collected, then infected into HepG2 cells and stable cells were obtained by screening with 50μg/ml zeocin and 1μg/ml puromycin and identified by westernbiotting with anti-TAP antibody. This established a stable basis for studying HBV-host interactome in the next step.