Expression of hepatitis B virus surface antigen P31 gene in yeast

The hepatitis B virus surface antigen (HBsAg) P31 gene has been expressed in yeast Saccharomyces cerevisiae. The gene products were shown to be glycoproteins with molecular sizes of 37,000 and 34,000 daltons (GP37 and GP34) containing polymerized albumin receptors. Successfully detecting these proteins depended on the extraction procedures. In the extract without protein denaturants and inhibitors, these products were degraded rapidly by proteases to yield smaller size derivatives lacking polymerized albumin receptors. As is the case in human serum-derived HBsAg, yeast HBsAg consisting of GP37 and GP34 was found to be particles or aggregates having a buoyant density of 1.2 g/cc; these particles bound to polymerized human serum albumin in species-specific manner.     

Expression of the hepatitis B virus surface,core and E antigen genes by stable rat and mouse cell lines

A recombinant plasmid containing four tandem head-to-tail copies of the HBV⁴ genome has been constructed and introduced into thymidine kinase-deficient mouse and rat fibroblast cells by co-transformation with the thymidine kinase gene from Herpes simplex virus as a selectable marker. Several mouse cell lines that synthesize HBsAg and four lines (three mouse and one rat) that synthesize both HBsAg and HBeAg were isolated. The rat line and at least one of the L cell lines that synthesizes HBeAg also produce small quantities of HBcAg. The rat line has many partial and 18 to 20 complete copies of the HBV genome integrated into high molecular weight DNA, whereas the L cells that produce HBeAg have only two to three complete copies of HBV genome. The kinetics of synthesis of HBsAg and HBeAg by the rat line are similar to that for HBsAg synthesis by two human hepatoma lines, and the HBsAg secreted by this line has similar biophysical characteristics to that found in human serum. The results suggest that the configuration of the recombinant molecule used may be an important prerequisite for the expression of HBcAg and HBeAg, and we speculate that the biogenesis of their messenger RNA may proceed via a precursor that is greater than the length of the HBV genome.     

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.     

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.     

Polypeptides of hepatitis B virus surface antigen produced by a hepatoma cell line.

The PLC/PRF/5 cell line derived from a human hepatoma produces hepatitis B surface antigen (HBsAg) in 22-nm particles of the same buoyant density as those found in the serum of infected patients. The HBsAg particles from this cell line were labeled with [35S]methionine and purified, and the polypeptides were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with those of serum-derived particles. The two major polypeptides of serum-derived HBsAg particles (p20 and p23) were found in the same relative amounts in the particles from the cell line. The three smallest of the five minor components observed in HBsAg particles from serum were present in particles from the cell line. These polypeptides (p31, p36, and p43), as well as p20 and p23, were precipitated with anti-HBs-containing serum. The two largest polypeptides of serum particles (p49 and p66) were not detected in particles from these cells. When the PLC/PRF/5 HBsAg particles were radiolabeled with tritiated sugars, p23, and not p20, was found to contain radioactivity, indicating that the pattern of polypeptide glycosylation is similar to that of serum HBsAg. None of the other possible gene products of hepatitis B virus was detected in the PLC/PRF/5-derived HBsAg particles, in the cells, or in the cell supernatants.     

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.     

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.     

Insulin suppresses hepatitis B surface antigen expression in human hepatoma cells

The human hepatoma Hep3B cells contain integrated hepatitis B viral genome and continually secret hepatitis B surface antigen (HBsAg). The production of HBsAg (but not alpha-fetoprotein) was suppressed by addition of low concentrations (0.1-1 nM) of insulin into serum-free medium. In addition, the suppression of HBsAg production by insulin was paralleled with the decrease in HBsAg mRNA abundance. Insulin also cause a rapid rate of disappearance of HBsAg mRNA (t 1/2, 2 h) in Hep3B cells. The Hep3B cells carry specific receptor with high affinity for insulin (Kd = 1.8 nM). The receptor showed an insulin-dependent protein tyrosine kinase activity. The half-maximal insulin concentration for the activation of the receptor kinase was about 5 nM. Only very high concentrations of insulin-like growth factor I and human proinsulin can compete for the insulin receptor binding and suppress HBsAg production, this suggests that insulin may act through its receptor binding to suppress HBsAg expression in human hepatoma Hep3B cells.     

Rearrangement of the surface antigen gene of hepatitis B virus integrated in the human hepatoma cell lines.

The rearrangement of integrated HBV DNA sequences in three different hepatoma cell lines, huH-1, huH-2, KG-55-T from Japanese patients, were studied by blot hybridization using whole HBV genome or a HBsAg or HBcAg DNA as a probe. The characteristic existence of multiple integration sites of HBV DNA sequences in each HindIII-restricted hepatoma cell DNA was revealed by the HBV genome probe. Detection of the isolated HBsAg gene in the HindIII fragment indicates that the integration of HBV DNA was not always related to the maintenance of the whole viral genome, and that movement of the HBsAg gene to another location occurred by rearrangement. On the other hand, the presence of the HBV DNA sequence without the intact HBcAg gene was shown in some of the HindIII fragments, when the HBcAg gene, probe was used, but a HindIII fragment, containing only the HBcAg gene, was not detected so far. The absence of the intact HBcAg gene suggests that the viral genome may lose a part of the HBcAg gene in the process of integration. This is consistent with recent findings of Ogston et al. (1982) that in Woodchuck hepatocellular carcinoma viral sequences are extensively rearranged.     

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     

Secretion of acetylcholinesterase by a mouse hepatocyte X rat liver cell hybrid culture

Summary A hybrid cell line (E-2) that secretes the enzyme acetylcholinesterase (AChE) has been prepared. The E-2 cell was the product of a fusion between primary mouse hepatocytes and a chemically transformed rat liver cell line (FRL), neither of which expresses AChE activity. The enzyme was determined to be AChE on the basis of its susceptibility to inhibition by BW284c51 but not by iso-OMPA, as well as its substrate specificity. Although the secreted enzyme was salt soluble and its activity not modified by the addition of the nonionic detergent, Triton X-100, the activity of the cellular enzyme (derived from homogenates of E-2 cells) was greatly enhanced in the presence of the detergent. This work was supported by funds from the Chemical Research and Development Center, Aberdeen Proving Ground, MD. The opinions and assertions are the private ones of the authors and are not to be construed as official. These experiments were conducted according to the principles set forth in the “Guide for the Care and Use of Experimental Animals,” DHEW Publ. No. (NIH) 78-23.     

Lipid composition of hepatitis B virus surface antigen particles and the particle-producing human hepatoma cell lines

More than 90% of lipids of hepatitis B virus surface antigen (HBsAg) particles produced by two human hepatoma cell lines (huGK-14 and PLC/PRF/5) were composed of phospholipids, with phosphatidylcholine being the dominant component, accounting for more than 80% of total membrane lipids. Analysis of subclass compositions of phospholipids of HBsAg particles and the host cell lines revealed that 1,2-diacyl glycerophosphocholine was preferentially incorporated into the membrane of the HBsAg particles, although both host cell lines contained extremely high concentrations (more than 60% of total phospholipids) of ether-linked phospholipids. Phospholipids of other hepatoma cell lines (HuH-7, Hep-G2, and huL-1) which were not associated with hepatitis B virus (HBV) infection, were composed mostly of 1,2-diacylglycerophospholipids. Activities of dihydroxyacetone-phosphate acyltransferase, which is known to be an obligatory enzyme in ether lipid biosynthesis, were found to be elevated by three- to fourfold in both huGK-14 and PLC/PRF/5 cells compared to those of other hepatoma cell lines. The results suggest a possible relationship between HBV-induced hepatocellular carcinogenesis and the drastic change in the metabolism of membrane phospholipids.