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)

Treatment of hepatitis B surface antigen (HBsAg) with either chloroform-methanol (2:1, v/v) or 50% 1,1',3,3'-tetramethylurea did not affect the morphological integrity of the particles (about 20 nm in diameter), although the major portion of lipids was released as indicated by their increased buoyant density in CsCl (1.27 g/cm3 as compared with 1.20 g/cm3 for intact HBsAg). The antigenicity and polypeptide composition of HBsAg was not altered by delipidation. The carbohydrate chains of HBsAg contain penultimate beta-D-galactosyl residues. HBsAg was cleaved by chymotrypsin into fragments which were smaller than intact HBsAg by two orders of magnitude and which contained both the a and d determinants.
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PMID:Properties of delipidated hepatitis B surface antigen (HBsAg) and preparation of its proteolytic cleavage fragments carrying HBsAg-specific antigenic determinants. 7 67

Various physico-chemical parameters have been studied in order to improve the production of hepatitis B virus pre-S2 antigen (middle surface antigen) by the methylotrophic yeast Hansenula polymorpha. Antigen production was done in two steps: first, production of cells on glycerol (Phase 1), followed by induction of antigen expression with methanol (Phase 2). Dense cultures of H. polymorpha, equivalent to 35-40 g/l (dry weight), were readily obtained in small fermenters using minimal medium containing glycerol as carbon source. Antigen expression in this minimal medium, after induction with methanol, was however, low and never exceeded 1.6 mg/l of culture. Antigen production was greatly enhanced by adding complex organic nitrogen sources along with methanol at induction time; yeast extract was the best of all the sources tested. In shake flasks, antigen production was proportional to yeast extract concentration up to 7% (w/v) yeast extract, it became clear the the nutritional conditions for good antigen expression were different from those for good biomass production. The effects of yeast extract were reproduced in small fermenters: antigen levels reached 8-9 mg/l in medium containing 6% (w/v) yeast extract during induction with methanol. The mechanisms of yeast extract's effects are still unknown but are probably nutritional. The recombinant H. polymorpha strain produced both periplasmic and intracellular antigen. The periplasmic antigen was shown to be present as 20-22-nm particles and was therefore immunogenic. Immunoblotting indicated that part of the pre-S2 antigen was present as a 24-kDa degradation product. These studies have led to a 140-fold increase in volumetric productivity of antigen and to a 4.6-fold increase in specific production.
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PMID:Fermentation study for the production of hepatitis B virus pre-S2 antigen by the methylotrophic yeast Hansenula polymorpha. 136 97

The recombinant gene for hepatitis B core antigen (HBcAg) was cloned and expressed, and the protein was purified from Escherichia coli cultures. Purified HBcAg was tested for the effects of various physical and chemical agents on its immunoreactivity by a paramagnetic particle-based enzyme immunoassay. Recombinant HBcAg retained its immunoreactivity when heated at 70 degrees C for 60 min but was inactivated at 85 degrees C in 10 min. It was stable between pHs 5 and 10.5 but not at pHs 2 and 13.5. Treatment with sodium dodecyl sulfate (SDS), ethanol, and methanol caused a significant loss in HBcAg reactivity. The proteolytic enzymes papain and bacterial protease (type VIII from Bacillus licheniformis) degraded HBcAg significantly, but trypsin and chymotrypsin did not. The effect of combined SDS and 2-mercaptoethanol on recombinant HBcAg was an immediate loss in immunoreactivity, followed by rapid recovery to about 50% of the initial level. This level was maintained for 24 to 48 h and was followed by an almost total loss of HBcAg in about 120 h.
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PMID:Stability of the recombinant hepatitis B core antigen. 162 88

An expression system has been developed for the methylotrophic yeast Hansenula polymorpha and used to co-express both the L (preS1-S2-S) and S hepatitis B surface antigens (HBsAg) under the control of strong methanol-inducible promoters derived from the methanol oxidase and from the formate dehydrogenase genes. A unique feature of this H. polymorpha expression system is the possibility of integrating up to 100 copies of an expression cassette via a multimeric integration mechanism. Several multimeric integrants containing various numbers of L and S expression cassettes were constructed to give a spectrum of strains characterized by different L to S ratios. The expression level of S antigen was 5-8% of the total soluble cell protein. Analysis by sucrose and CsCl density gradient centrifugation and by particle-specific immunoassays demonstrated that the synthesized HBsAg spontaneously assembled into composite subviral particles containing both S and L proteins. Only a minor portion of the L protein was found to be glycosylated. These H polymorpha-derived composite particles can be used for the production of a hepatitis B virus vaccine with the potential for improved immunogenicity due to the presence of a wider spectrum of epitopes and negligible glycosylation.
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PMID:Simultaneous expression of the S and L surface antigens of hepatitis B, and formation of mixed particles in the methylotrophic yeast, Hansenula polymorpha. 189 10

The methylotrophic yeast, Hansenula polymorpha, has been developed as a host system for the synthesis of heterologous proteins. The middle surface antigen of hepatitis B virus (preS2-HBsAg) has been synthesized under the control of a methanol-regulated promoter derived from the methanol oxidase-encoding gene. The synthesized preS2-HBsAg protein was found to be secreted outside the cell membrane into the periplasm and further excreted into the culture medium following permeabilization of the cell wall with beta-1,3-glucanase (beta Glu). Cell cultures treated with beta Glu were able to continuously synthesize and secrete 22-nm particles of preS2-HBsAg into the medium for several days. The overall yield of antigen from treated cultures was found to be over threefold greater than that of untreated controls. The observation that complex supramolecular structures, such as the 22-nm particles of preS2-HBsAg, can be secreted by H. polymorpha and released into the medium, suggests the potential for these yeasts to be an alternative secretory host.
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PMID:Synthesis and secretion of hepatitis B middle surface antigen by the methylotrophic yeast Hansenula polymorpha. 251 17

Non-degraded thimerosal was determined in the presence of its decomposition products by directly assaying recombinant hepatitis B vaccine using a reversed-phase liquid chromatographic method. Methanol-water-orthophosphoric acid (65:35:0.9, v/v/v) was used as the eluent. Salicylic acid was employed as an internal standard. The calibration graph was linear (r = 0.99995) up to 2.5 micrograms of thimerosal. Interference from aluminium hydroxide was eliminated by centrifugation. Good stability of thimerosal in the hepatitis B vaccine was demonstrated. The results obtained were in agreement with the recently proposed mechanism of degradation.
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PMID:Reversed-phase high-performance liquid chromatographic study of thimerosal stability in Cuban recombinant hepatitis B vaccine. 900 43

High-level expression and efficient assembly of hepatitis B surface Antigen (HBsAg) has been reported in Pichia pastoris by integrating a single copy of the HBsAg gene under control of the AOX1 promoter. To fully utilize the expression potential of the P. pastoris expression system, we investigated the influence of gene copy number on the expression of HBsAg in this yeast. A panel of Pichia clones carrying progressively increasing copies of the heterologous gene expression cassette was created using an in vitro multimerization approach. Using this strategy, constructs containing up to a maximum of eight direct repeats of the HBsAg-expressing cassettes could be created. These expression cassettes were targeted for integration into the genome of the host strain GS115 with simultaneous elimination of the resident AOX1 gene. Deletion of the AOX1 gene was intended to create Mut(s) (methanol utilization slow) transformants that are known to have an increased ability to generate HBsAg in particulate form. A systematic investigation of the resultant clones demonstrated that the increase in copy number results in a proportional elevation in the steady-state levels of the HBsAg-specific mRNA, which in turn is closely paralleled by a corresponding increase in the total levels of the HBsAg protein. Virtually all the recombinant protein in the soluble fraction was present in the particulate form based on particle-specific ELISA and sedimentation behavior. Further, our studies also revealed the continued physical and functional integrity of the HBsAg-expressing cassettes during the course of an extended induction phase spanning 6 days.
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PMID:Effect of copy number on the expression levels of hepatitis B surface antigen in the methylotrophic yeast Pichia pastoris. 1116 89

High-level expression and efficient assembly of Hepatitis B surface Antigen (HBsAg) particles have been reported in Pichia pastoris by integrating a single copy of the HBsAg gene under the control of the alcohol oxidase (AOX1) promoter. However, the time taken to reach peak product concentration is usually very long ( approximately 240 h). In this paper, we describe the expression of HBsAg in P. pastoris using the recently described glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter. Unlike the previously described AOX1 promoter based system (in which biomass is generated first followed by methanol-induced antigen production), biomass generation and antigen production occur simultaneously in medium containing glycerol or glucose. Maximal levels of HBsAg expression in case of the single copy AOX1 integrant (attained after 6 days of induction) exceeded the levels of antigen produced by the single copy GAP integrant. However, this was offset by continuous antigen production by the GAP clone. In an attempt to further enhance antigen production levels of the GAP clones, we isolated multicopy Pichia integrants containing up to four copies of the GAP promoter-driven constitutive expression cassette using the Zeocin screening procedure. The data demonstrated a direct correlation between the gene dosage and the levels of HBsAg expressed by the GAP clones. The effect of copy number was additive and the four copy clone resulted in about four-fold higher yield of HBsAg. The majority of HBsAg produced in the constitutive expression system was found to be of particulate form, based on sedimentation behaviour and particle-specific ELISA, suggesting that it has the potential to serve as an effective immunogen. These particles were sensitive to thiol reagents. We also explored the possibility of secreting the GAP expressed HBsAg in P. pastoris. In-frame fusion of the Saccharomyces cerevisiae alpha-factor secretion signal under the constitutive GAP promoter resulted in secretion of approximately 20 nm HBsAg particles as evidenced by electron microscopy. However, the levels of secreted HBsAg particles were very low, presumably due to the inherent hydrophobicity of the HBsAg molecule and the consequent propensity for membrane association. Our studies show that secretion is not a good strategy for expression of HBsAg in P. pastoris. The data also suggests that intracellular production of HBsAg under the GAP promoter using multicopy expression cassettes can indeed serve as an effective alternative to the AOX1 promoter. Further, the GAP promoter based system obviates the need to use and extensively monitor methanol during recombinant antigen production. Finally, this constitutive system has the potential for continuous culture wherein several batches of recombinant protein-containing biomass can be harvested from a single initial fermentation.
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PMID:Expression of hepatitis B surface antigen in the methylotrophic yeast Pichia pastoris using the GAP promoter. 1137 62

The methylotrophic yeast, Pichia pastoris, has been used as a host to express the envelope protein (Den2E) of dengue type 2 virus (NGC strain) as a chimera with hepatitis B surface antigen (HBsAg): a protein known to self assemble into virus-like particles (VLPs) and to be efficiently expressed in P. pastoris. The Den2E gene used in this study is a truncated version encoding the first 395 amino acid (aa) residues of the mature Den2E protein; the HBsAg gene encodes the full length 226 aa HBsAg protein. Two in-frame gene fusions were constructed for intracellular expression in P. pastoris. The first one contains the HBsAg gene as the 5' partner and the Den2E gene as the 3'partner (HBsAg-Den2E). In the second one, the relative positions of the two partners of the gene fusion were reversed to create the hybrid Den2E-HBsAg gene. These fusion genes were integrated into the genome of P. pastoris under the control of the methanol-inducible alcohol oxidase (AOX1) promoter. Of the two fusions, the Den2E-HBsAg gene was expressed at higher levels in P. pastoris based on Northern analysis. The hybrid protein ( approximately 68 kDa) expressed by this clone was purified to near homogeneity using a combination of acid precipitation, hydrophobic interaction, and immunoaffinity chromatographic steps. Final purification achieved was approximately 1400-fold with a yield of approximately 26%. The chimeric protein was found to possess the ability to assemble into high molecular weight aggregates (akin to HBsAg particles). The recombinant fusion protein eluted close to the void volume of a Sepharose CL-4B column indicating its macromolecular nature. On a CsCl density gradient the recombinant fusion protein sedimented to a position very similar to that of HBsAg VLPs. The hybrid protein is recognized by the two neutralizing monoclonals against the two components of the chimeric protein.
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PMID:Expression and purification of Dengue virus type 2 envelope protein as a fusion with hepatitis B surface antigen in Pichia pastoris. 1157 Aug 49

A new class of 5-(1-cyanamido-2-haloethyl)-2'-deoxyuridines (4-6) and arabinouridines (7, 8) were synthesized by the regiospecific addition of halogenocyanamides (X-NHCN) to the 5-vinyl substituent of the respective 5-vinyl-2'-deoxyuridine (2) and 2'-arabinouridine (3). Reaction of 2 with sodium azide, ceric ammonium nitrate, and acetonitrile-methanol or water afforded the 5-(1-hydroxy-2-azidoethyl)-(10) and 5-(1-methoxy-2-azidoethyl)-2'-deoxyuridines (11). In vitro antiviral activities against HSV-1-TK(+) (KOS and E-377), HSV-1-TK(-), HSV-2, VZV, HCMV, and DHBV were determined. Of the newly synthesized compounds, 5-(1-cyanamido-2-iodoethyl)-2'-deoxyuridine (6) exhibited the most potent anti-HSV-1 activity, which was equipotent to acyclovir and superior to 5-ethyl-2'-deoxyuridine (EDU). In addition, it was significantly inhibitory for thymidine kinase deficient strain of HSV-1 (EC(50) = 2.3-15.3 microM). The 5-(1-cyanamido-2-haloethyl)-2'-deoxyuridines (4-6) all were approximately equipotent against HSV-2 and were approximately 1.5- and 15-fold less inhibitory for HSV-2 than EDU and acyclovir, respectively. Compounds 4-6 were all inactive against HCMV but exhibited appreciable antiviral activity against VZV. Their anti-VZV activity was similar or higher to that of EDU and approximately 5-12-fold lower than that of acyclovir. The 5-(1-cyanamido-2-haloethyl)-(7,8) analogues of arabinouridine were moderately inhibitory for VZV and HSV-1 (strain KOS), whereas compounds 10 and 11 were inactive against herpes viruses. Compounds 5 and 6 also demonstrated modest anti-hepatitis B virus activity against DHBV (EC(50) = 19.9-23.6 microM). Interestingly, the related 5-(1-azido-2-bromoethyl)-2'-deoxyuridine (1n) analogue proved to be markedly inhibitory to DHBV replication (EC(50) = 2.6-6.6 microM). All compounds investigated exhibited low host cell toxicity to several stationary and proliferating host cell lines as well as mitogen-stimulated proliferating human T lymphocytes.
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PMID:Synthesis and antiviral activity of novel 5-(1-cyanamido-2-haloethyl) and 5-(1-hydroxy(or methoxy)-2-azidoethyl) analogues of uracil nucleosides. 1158 57


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