Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0019163 (hepatitis B)
 38,309 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A fraction of the large surface protein (L) of duck hepatitis B virus (DHBV) is phosphorylated at serine or threonine residues (E. Grgacic & D. Anderson, Journal of Virology 68, 7344-7350, 1994). We now report the identification of phosphorylation sites in DHBV L protein. Using site-directed mutagenesis, we have identified serine-118 (S118) as the major phosphorylation site, accepting approximately 64% of the total phosphate groups incorporated in L, and resulting in retarded migration of phosphorylated L in SDS-PAGE. Proline-119 is indispensable for S118 phosphorylation. Mutation of other serine/threonine residues which are followed by prolines (T79, T89, S117 and T155) together with S118 further reduced phosphorylation to around 19% of wild-type. Non-equilibrium pH gel electrophoresis (NEPHGE) and SDS-PAGE of 33P-labelled L protein revealed two phosphorylated L species, while protein with the S118 to alanine mutation was detected as only one labelled species, consistent with multiple phosphorylations in wild-type L. Together, these results demonstrate that serine 118 is the major phosphorylation site for a proline-directed kinase, and that a proportion of L molecules are additionally phosphorylated at one of a number of secondary sites. DHBV mutants encoding L proteins with minimal phosphorylation (alanine mutants) or mimicking constitutive phosphorylation (aspartic acid mutants) remained infectious both in cell culture and in ducks, demonstrating that L phosphorylation may play only a minor role in DHBV replication.
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PMID:Normal phosphorylation of duck hepatitis B virus L protein is dispensable for infectivity. 982 Jan 50

The physicochemical properties of recombinant hepatitis B surface antigen (r-HBsAg), which was expressed in C127 mammalian cell were studied. Using roller bottle culture in DMEM supplemented with fetal bovine serum, 10-15 mg/L of r-HBsAg was produced with about 31% of purification yield. The purity of r-HBsAg by HPLC was 99.8% and electron microscopic examination showed homogeneous spherical particle with 22 nm in diameter, a morphological characteristic of HBsAg. The density of r-HBsAg by CsCl density gradient method was 1.19 g/ml and the isoelectric point by Mono P HR 5/20 column was 4.6. The analysis of subunit protein pattern using SDS-PAGE followed by scanning densitometry gave 81.3% of S protein and 18.7% of pre-S protein. Fluorophore-assisted-carbohydrate-electrophoresis analysis showed the relative amount of carbohydrate to protein was 1.7% and its major component was N-acetyl glucosamine, which was about 39% of total carbohydrate. The relative amount of lipid to protein determined by vanillin phosphoric acid method was 32.5% and its major component was phospholipid, which was about 70% of total lipid. The physicochemical properties of C127 mammalian cell-derived r-HBsAg are similar to those of p-HBsAg, suggesting that the r-HBsAg can be used in developing a new preventive vaccine against hepatitis B.
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PMID:Physicochemical properties of recombinant hepatitis B surface antigen expressed in mammalian cell (C127). 987 88

Besides the three essential genes encoding the envelope, core and polymerase proteins, all mammalian hepadnaviruses examined to date contain a fourth gene which is referred to as the x-gene. This gene is believed to encode a transcriptional transactivator which positively regulates viral gene expression. Attempts to detect X-protein in vivo or in tissue culture lead to varying results. Whereas some groups could detect a protein of the expected size, other groups did not. To establish optimal conditions for the isolation of the human hepatitis B virus X-protein, we introduced a recognition site for protein kinase A into the x-gene. Upon phosphorylation with radioactive ATP, this modified X-protein can be detected with very high specificity and sensitivity. Tissue culture experiments showed that X-protein expressed from a cytomegalovirus-driven plasmid is not soluble in non-ionic detergent but rather has to be extracted from the cell pellet by boiling with SDS at a slightly alkaline pH. This method was then used to examine the organs of several transgenic mouse lines which expressed the modified x-gene under control of the authentic promoter. The data show that expression of the x-gene and subsequent biosynthesis of the X-protein is not tissue-specific but rather can occur in most organs.
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PMID:Detection of the human hepatitis B virus X-protein in transgenic mice after radioactive labelling at a newly introduced phosphorylation site. 1050 7

An assay for the detection of yeast (Saccharomyces cerevisiae) protease activity, using partially purified yeast-derived recombinant hepatitis B surface antigen (rHBsAg) as substrate, was developed to monitor proteolysis of rHBsAg that may occur through fermentation and isolation. The method consists of incubating small amounts of yeast lysate (protease source) with the substrate at 35 degrees C for about 16 h. Substrate proteolysis is assessed by subjecting the incubation mixtures to SDS-PAGE followed by silver-staining. The type of protease responsible for particular cleavages can be identified by treating the yeast lysates with specific protease inhibitors prior to incubation with substrate. The treatment of lysates with PMSF indicated that while many lysates possessed only serine protease activity (Protease B), some possessed proteolytic activity that could not be quenched with high levels of PMSF or other serine protease inhibitors. The use of the aspartyl protease inhibitor Pepstatin A in conjunction with PMSF virtually eliminated all proteolytic activity in these lysates, indicating that an aspartyl protease (Protease A) is expressed under some fermentation conditions. The relative amount of each protease in a lysate can be determined semiquantitatively by scanning the SDS gels densitometrically and plotting the ratio of degradates to intact antigen in the presence and absence of protease inhibitors. This method was used successfully to monitor the time-dependent expression of these proteases throughout production-scale fermentations. The impact of fermentation and purification changes on those proteases specifically responsible for the rHBsAg degradation can be easily evaluated.
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PMID:Identification and monitoring of protease activity in recombinant Saccharomyces cerevisiae. 1059 23

Fetal calf serum (FCS) was depleted of its immunoglobulin G (IgG) in a rapid procedure using protein G affinity chromatography. 20 ml of FCS was depleted of its IgG in less than 80 min by applying 5 ml of FCS to a 1 ml HiTrap protein G Sepharose column followed by appropriate elution. Various concentrations of IgG-depleted FCS (G-FCS) were used in RPMI-1640 medium to grow the mouse hybridoma cell lines CAy-G (anti-HBs IgG1 mAb producing hybridoma cell) and CAy-M (anti-HBs IgM mAb producing hybridoma cell), which secreted hepatitis B virus surface antigen (HBsAg)-reactive IgG1 and IgM monoclonal antibodies (mAbs), respectively. Antibody production and cell growth were used as indices to compare the efficacy of RPMI/G-FCS with that of RPMI/FCS and serum/protein-free Hybri Max (Sigma, MO, USA) hybridoma medium. MAb production and cell growth of CAy-G and CAy-M hybridoma cell lines in RPMI/G-FCS were similar to culture in RPMI/FCS and significantly better than culture in Hybri Max. We found that G-FCS was superior to whole FCS as a culture supplement for the purification of IgG1 mAbs. IgG1 mAbs were isolated in a single-step procedure using protein G affinity chromatography, from the supernatant of CAy-G hybridoma cells cultured in RPMI/10% G-FCS (RPMI-1640 medium supplemented with 10% G-FCS). SDS-PAGE analysis revealed that the purity of IgG isolated from the supernatant of CAy-G cells cultured in RPMI/10% G-FCS was more than 99%.
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PMID:Development of a rapid, single-step procedure using protein G affinity chromatography to deplete fetal calf serum of its IgG and to isolate murine IgG1 monoclonal antibodies from supernatants of hybridoma cells. 1064 58

The affinity of natural antibody (Ka = 8 x 10(6) M(-1)) recognizing preS1 of hepatitis B virus (HBV) was improved by replacing the heavy (H) chain gene with repertoires of VH genes, obtained from two nonimmunized donors. Two separate clones, 1C2 and 1E4, showed affinities of 2.3 x 10(7) and 5.2 x 10(7) M(-1), which were increased by factors of 2.8 and 6.5, respectively, compared to the parental clone. Recombinant scFvs (rscFvs) were expressed as fusion protein with minor coat protein, pIII, and secreted into medium after 3 h of induction with 1 mM IPTG. The expression level of functional rscFv capable of binding to preS1 reached a peak after 6-10 h (1C2) and 8-10 h (1E4) of IPTG induction, and afterwards decreased gradually. In order to achieve the overexpression of rscFv in E. coli, gene encoding scFv of 1C2 or 1E4 was inserted into pRSET vector. RscFvs were overexpressed as cytoplasmic inclusion bodies in E. coli BL 21 strain, which were denatured and carefully refolded using a continuous dialysis system. The purified recombinant fragments were pure when analyzed by SDS-PAGE and had the predicted size of 34 kDa. Clone 1E4 used the heavy chain gene belonging to family VII and subgroup III. Chain shuffling offers an alternative to random point mutation for affinity maturation of human antibody in vitro.
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PMID:Affinity maturation of natural antibody using a chain shuffling technique and the expression of recombinant antibodies in Escherichia coli. 1096 2

Hepatitis B virus infection is primarily mediated by the interaction of the preS region of the viral envelope protein with its still unknown cellular receptor. Using recombinantly expressed preS proteins, the distribution of preS-binding receptors on cell lines from extrahepatic origins was determined by immunofluorescence and flow cytometry. In contrast to human liver cell lines, most cell lines from extrahepatic origins did not bind preS proteins. Nevertheless, exceptions were found in the bone marrow-derived cell line, KG-1, and the osteogenic sarcoma cell line SaOS-2, as well as in the previously reported EBV-transformed B-cell line, Wa. To determine the biochemical nature of these receptors, Wa-cells were cell surface biotinylated and the preS-binding receptors were isolated by immunoprecipitation. A specific band with a molecular weight of approximately 30 kDa was identified in a SDS-polyacrylamide gel, which further characterization is expected to provide clues regarding the infection mechanism of HBV in hepatic- and extra-hepatic cells.
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PMID:Detection of cellular receptors specific for the hepatitis B virus preS surface protein on cell lines of extrahepatic origin. 1102 70

Woodchuck hepatitis virus (WHV) is closely related to the human hepatitis B virus (HBV) and infection of woodchucks with WHV creates a useful model for studies of immunity, pathogenesis and therapy of HBV infection. To increase the usefulness of this model, monoclonal antibodies were raised to woodchuck hepatitis surface antigen (WHsAg) and one of these antibodies was used to purify the antigen by affinity chromatography from serum, a simpler and quicker method of purification than the current ultracentrifugation methods. The bands found by SDS-polyacrylamide gel electophoresis of WHsAg were the major 25 and 29 kilodalton (kDa) bands and a triplet of 45, 51 and 55 kDa which are thought to be the glycosylated and unglycosylated middle and large WHsAg. Both the antibody and the antigen are valuable reagents for the study of WHV infection.
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PMID:Purification of woodchuck hepatitis surface antigen using a monoclonal antibody raised against the antigen. 1131 48

The preS1 of hepatitis B virus (HBV) is located at the outermost part of the envelope protein and possesses several functionally important regions such as hepatocyte receptor-binding site and virus-neutralizing epitopes. As the first step to understand the structure-function relationship for the preS1 antigen, we have purified the preS1 and performed its structural characterization by circular dichroism (CD) spectroscopy. The preS1 was purified to near homogeneity from bacterially expressed glutathione S-transferase (GST)-preS1 fusion protein by two-step purification, affinity chromatography on glutathione-agarose column, and cation-exchange chromatography on Mono S column. The CD analysis showed that the purified preS1, which was largely unstructured in aqueous solution, acquired a significant (16%) alpha-helical structure when analyzed in 50% trifluoroethanol or 20 mM SDS. The results suggest that the preS1 assumes a mainly unstructured conformation and may form induced secondary structures upon binding to target proteins or under hydrophobic environment.
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PMID:Purification and structural analysis of the hepatitis B virus preS1 expressed from Escherichia coli. 1140 32

Expression plasmids pGEXSI and pGEXSII containing one copy and two orderly joined copies of PreS1(21--47 aa) DNA fragment, respectively, were constructed. GST-PreS1(21--47 aa) and GST-2xPreS1(21--47 aa) fusion proteins were highly expressed in E.Coli TG1, induced by IPTG. The expression level of GST-PreS1(21--47 aa) was about 30% of total soluble proteins in the lysate of expression bacteria, and GST-2xPreS1(21--47 aa) was about 15% of total soluble proteins, asestimated by SDS-PAGE. 50 mg GST-PreS1(21--47 aa) or 20 mg GST-2xPreS1(21--47 aa) with purity over 90% was obtained, respectively, from 1 L culture by using affinity chromatography of glutathione-Sepharose 4B. Direct ELISA results showed that antigenicity of GST-2xPreS1(21--47 aa) was better than GST-PreS1(21--47 aa) and synthetic peptide. Using GST-2xPreS1(21--47 aa) as coated antigen, a sensitive indirect ELISA for detection of anti-PreS1(21--47 aa) antibody, based on protein A-biotin and streptavidin-HRP, was established. The results from 99 sera samples of hepatitis B patients showed that anti-PreS1(21--47 aa) antibody was detected in nearly half of acute hepatitis B patients during recovery, but it was detected only in a few chronic hepatitis patients. Clinical follow-up study suggested that appearance of anti-PreS1(21--47 aa) was related to the course of the disease and recovery of patients. Detection system established in the study is promising for clinical application.
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PMID:Expression, Purification and Preliminary Clinical Use of Recombinant HBsAg GST-PreS1(21--47 aa) Fusion Proteins. 1204 Apr 9


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