Expression of hepatitis B virus surface antigen P31 gene in Escherichia coli

A hepatitis B virus surface antigen (HBsAg) P31-coding DNA was constructed from a DNA fragment of the plasmid pHBr330 containing the entire hepatitis B virus (HBV) adr DNA and a chemically synthesized adaptor. The P31 gene was inserted into an expression vector, pTRP771, having an Escherichia coli tryptophan operon (trp) promoter to give a recombinant plasmid pTRP P31-R. The distance between the Shine-Dalgarno (SD) sequence and the initiation codon of P31 gene was adjusted to 9 bp. The expression level of HBsAg by E. coli 294[pTRP P31-R] was significantly elevated, in contrast to that of HBsAg by E. coli 294[pTRP SS-6]. Western blotting analysis has shown that E. coli[pTRP P31-R] synthesizes a specific polypeptide P31 of about 31 kDal, which reacts with anti-HBsAg antibody. The binding studies with polyalbumins from various species have also suggested that HBsAg P31 specifically binds to polymerized human serum albumin.     

Synthesis in yeast of hepatitis B virus surface antigen modified P31 particles by gene modification

The pre-S2 portion of hepatitis B virus surface antigen P31 gene was modified to make gene products resistant to trypsin-like proteases in Saccharomyces cerevisiae. The coding sequence for 6 amino acids (Ser44 - Thr49) including Arg48 was removed, and the altered gene was inserted into an expression vector. The modified HBsAg P31 (M-P31c) gene products, consisting of GP37 and GP34, formed particles having both HBsAg antigenicity and polymerized-albumin receptor activity. Since the M-P31c particles can elicite two kinds of protective antibodies against hepatitis B virus, anti-S and anti-pre-S2 antibodies, the M-P31c particles are expected to be potentially effective to S-nonresponders.     

Efficient expression of genetically engineered hepatitis B virus surface antigen P31 proteins in yeast

We have constructed plasmids that express modified hepatitis B virus surface antigen (HBsAg) P31-coding genes (M-P31c, d, e, f, and i) having various genetically engineered pre-S2 regions. The plasmids contain the GAPDH (gene coding for glyceraldehyde-3-phosphate dehydrogenase) promoter and the PGK (gene coding for 3-phosphoglycerate kinase) terminator, both isolated from sake brewing yeast, Saccharomyces cerevisiae Kyokai III. Expression levels of the modified HBsAg P31 proteins in yeast are greatly increased from 0.4% to 11.7% of total cell protein. However, the specific mRNAs are expressed at equal levels and the degradation rates of the modified P31 proteins do not vary significantly. Therefore, we considered that different expression levels of the modified P31 proteins are attributed to the changes of the post-translational efficiency. And it was suggested that the conformational stability of the N-terminal peptide (Met-1-Phe-46) in the endoplasmic reticulum membrane determines the expression level of modified P31 proteins.     

Expression of hepatitis B surface antigen by yeast actin gene promoter

The promoter sequence (from position −500 to −21) of the yeast actin gene was subcloned into the multiple cloning site of pUC12 to generate a new recombinant plasmid pYAP12. The actin gene promoter can therefore be readily excised from pYAP12 by several restriction enzymes and subsequently placed upstream of the desired protein coding sequence. Results of expression of the hepatitis B surface antigen controlled by the actin gene promoter of pYAP12 in yeast suggest it is a strong functional promoter. To our knowledge, this is the first demonstration of the potential application of the yeast actin gene promoter for expression of foreign proteins in yeast.     

Expression of hepatitis B virus surface antigen gene in Escherichia coli

Direct expression of hepatitis B virus (HBV) surface antigen (HBsAg) gene under the control of the Escherichia coli tryptophan operon (trp) promoter has been achieved. Synthesis of HBsAg (both complete and lacking its N-terminal segment) as a part of hybrid proteins with the N-terminal portion coded by genes cat, kan or bla is controlled by the appropriate promoters, as well as by the trp promoter. The highest levels of expression, including those for direct synthesis of HBsAg, provide the accumulation of about 10⁵ polypeptide molecules per bacterial cell.     

Characteristics of hepatitis B surface antigen produced in yeast

We have constructed an expression plasmid for regulated expression of the hepatitis B surface antigen gene in yeast using promoter of the yeast Pho5 gene. In the yeast transformants, the monomeric HBsAg (22K dalton) was estimated to constitute approximately 3% of the total proteins. On extraction, the HBsAg was found to have a buoyant density of 1.18 g/ml and an Sw.20 value of 54. Electron microscopy revealed particles of heterogeneous size ranging from 18-28 nm. When the yeast HBsAg was used to immunize guinea pigs, the anti-HBsAg antibodies produced could react with human serum HBsAg.     

Expression of the hepatitis B virus surface antigen in Drosophila S2 cells

Drosophila melanogaster S2 cells were transfected with a plasmid vector (pAcHBsAgHy) containing the S gene, coding for the hepatitis B virus surface antigen (HBsAg), under control of the constitutive drosophila actin promoter (pAc), and the hygromycin B (Hy) selection gene. The vector was introduced into Schneider 2 (S2) Drosophila cells by DNA transfection and a cell population (S2AcHBsAgHy) was selected by its resistance to hygromycin B. The pAcHBsAgHy vector integrated in transfected S2 cell genome and approximately 1,000 copies per cell were found in a higher HBsAg producer cell subpopulation. The HBsAg production varied in different subpopulations, but did not when a given subpopulation was cultivated in different culture flasks. Higher HBsAg expression was found in S2AcHBsAgHy cells cultivated in Insect Xpress medium (13.5 μg/1E7 cells) and SFX medium (7 μg/1E7 cells) in comparison to SF900II medium (0.6 μg/1E7 cells). An increase of HBsAg was observed in culture maintained under hygromycin selection pressure. Data presented in the paper show that S2AcHBsAgHy cells produce efficiently the HBsAg which is mainly found in the cell supernatant, suggesting that HBsAg is secreted from the cells. The data also show that our approach using the Drosophila expression system is suitable for the preparation of other viral protein preparation.     

Fermentation of recombinant yeast producing hepatitis B surface antigen

Summary Fermentations were performed to determine parameters affecting the expression of hepatitis B surface antigen (HBsAg) in the yeastSaccharomyces cerevisiae containing the HBsAg gene. These studies emphasized inereasing both the relative abundance (HBsAg: cell mass) and total production of HBsAg. Specific activity was increased 70-fold when cells were grown in shake flasks containing nonselective rather than selective medium. The addition of adenine, ammonium sulfate or glucose to the complex medium reduced the production of antigen. Results similar to those achieved in shake flasks were obtained when the growth was performed in fermenters. A nutrient addition system was employed to increase the production of cells and HBsAg. The addition of glucose to the culture medium increased cell mass 6-fold but decreased the production of antigen. This imbalance was corrected by supplementing the glucose with complex nutrients.     

Expression of hepatitis B virus large envelope polypeptide inhibits hepatitis B surface antigen secretion in transgenic mice.

The outer membrane of the hepatitis B virus consists of host lipid and the hepatitis B virus major (p25, gp28), middle (gp33, gp36), and large (p39, gp42) envelope polypeptides. These polypeptides are encoded by a large open reading frame that contains three in-phase translation start codons and a shared termination signal. The influence of the large envelope polypeptide on the secretion of hepatitis B surface antigen (HBsAg) subviral particles in transgenic mice was examined. The major polypeptide is the dominant structural component of the HBsAg particles, which are readily secreted into the blood. A relative increase in production of the large envelope polypeptide compared with that of the major envelope polypeptide led to profound reduction of the HBsAg concentration in serum as a result of accumulation of both envelope polypeptides in a relatively insoluble compartment within the cell. We conclude that inhibition of HBsAg secretion is related to a hitherto unknown property of the pre-S-containing domain of the large envelope polypeptide.     

Expression of the hepatitis B virus surface antigen gene by rat hepatocyte X mouse hepatoma hybrid cells

The expression of the S gene of hepatitis B virus has been studied in the somatic hybrid cells resulting from the fusion between rat hepatocytes in primary culture and cells of the mouse hepatoma line BWTG3, and in the parental line BWTG3. The DNA of the S gene inserted into the plasmids pAC Tk+ and pNY4 has been co-transfected into these cells with a plasmid DNA bearing a resistance gene to aminoglycoside. The level of expression of the S gene among the co-transfected resistant clones was estimated by radioimmunoassay. The results show that a high number of the co-transfected cellular hybrid clones express the S gene, whereas it is found, by contrast, that the S gene is poorly expressed in the mouse hepatoma cells. The level of expression of the S gene (as the amount of HBs Ag synthesized) is high in the hybrid clones and the synthesis of the HBs antigen is stable in time. These observations suggest for the first time in cell cultures in vitro, the role which is probably played by the normal hepatocyte genome in the expression of the S gene of HBV.     

Expression of hepatitis B surface antigen in the methylotrophic yeast Pichia pastoris using the GAP promoter

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.     

Expression of hepatitis B virus middle and large surface antigen genes in Saccharomyces cerevisiae.

The hepatitis B virus genome carries the surface antigen (SAg) gene and an open reading frame that encodes two SAg-related polypeptides: SAg with a 55-amino-acid N-terminal extension polypeptide and SAg with a 174-amino-acid N-terminal extension polypeptide. These are termed middle S and large S, respectively. These polypeptides or their glycosylated derivatives have been detected in Dane particles, but their chemical and biological properties have remained largely unknown because of their limited availability. We attempted to produce these proteins in Saccharomyces cerevisiae by placing the coding regions under the control of the promoter of the yeast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. Yeast cells carrying middle S and large S coding sequences produced 33,000- and 42,000-dalton products, respectively, each of which reacted with anti-S antibody and bound to polymerized human serum albumin, in accordance with the known properties of pre-S proteins from particles in human sera (K. H. Heermann, U. Goldmann, W. Schwartz, T. Seyffarth, H. Baumgarten, and W. H. Gerlich, J. Virol. 52:396-402, 1984; A. Machida, S. Kishimoto, H. Ohnuma, K. Baba, Y. Ito, H. Miyamoto, G. Funatsu, K. Oda, S. Usuda, S. Togami, T. Nakamura, Y. Miyakawa, and M. Mayumi, Gastroenterology 86:910-918, 1984). The middle S polypeptide is glycosylated and can be assembled into particles whose size and density are similar to those of SAg. However, this polypeptide was highly susceptible to proteolytic degradation into 29,000- and 26,000-dalton polypeptides, of which only the former retained the binding activity to polymerized albumin. The large S polypeptides are nonglycosylated, relatively stable, and do not seem to assemble into particles by themselves.     

Expression of the hepatitis B virus surface antigen in mammalian cells using an Epstein-Barr-virus-derived vector

 The hepatitis B virus surface antigen (HBsAg) gene, under control of the inducible mouse metallothionein I gene promoter, was inserted in an expression vector based on the Epstein-Barr virus (EBV). This vector was introduced into human cells by DNA transfection and clones were selected for their resistance to hygromycin B. The recombinant EBV vector replicates efficiently as an episome in human cells and approximately six copies per cell were found in one clone of hygromycin-B-resistant cells. These cells produce high levels of HBsAg in the presence of metals. The protein is mainly found in the cell medium, suggesting that the HBsAg is secreted from the cells. Received: 25 February 1996 / Received revision: 21 June 1996 / Accepted: 15 July 1996     

Production of marker-free plants expressing the gene of the hepatitis B virus surface antigen

The pBM plasmid, carrying the gene of hepatitis B virus surface antigen (HBsAg) and free of any selection markers of antibiotic or herbicide resistance, was constructed for genetic transformation of plants. A method for screening transformed plant seedlings on nonselective media was developed. Enzyme immunoassay was used for selecting transgenic plants with HBsAg gene among the produced regenerants; this method provides for a high sensitivity detection of HBsAg in plant extracts. Tobacco and tomato transgenic lines synthesizing this antigen at a level of 0.01–0.05% of the total soluble protein were obtained. The achieved level of HBsAg synthesis is sufficient for preclinical trials of the produced plants as a new generation safe edible vaccine. The developed method for selecting transformants can be used for producing safe plants free of selection markers.