Production and secretion in Escherichia coli of hepatitis B virus pre-S2 antigen as fusion proteins with beta-lactamase.

The diagnostically important surface antigen pre-S2 of hepatitis B virus was produced in large amounts in the periplasmic space of Escherichia coli. The DNA fragments (pre-S2) coding the pre-S2 antigen were tandemly duplicated or triplicated and ligated in the same reading frame to a fragment containing the promoter and the signal sequence of the alkaline phosphatase-coding gene (phoA) of E. coli. Further, a DNA fragment (bla) coding mature beta-lactamase was joined to the region coding the C terminus of the pre-S2 repeat to stabilize the gene product. Upon induction of the phoA-(pre-S2)3-bla fusion gene, the fusion protein was produced at up to 30% of the total cellular protein. Fractionation of the cellular components and trypsin accessibility of the product showed that the antigen was secreted in the periplasm and formed inclusion bodies there. The signal sequence of alkaline phosphatase was found to be correctly processed in E. coli.     

Efficient expression and characterization of hepatitis B virus preS2 antigen in Escherichia coli

The complete (encoding 55 amino acids, aa) or partial (encoding aa 1–26) preS2 region gene of hepatitis B virus (HBV) was fused to the 3-end of glutathion-S-transferase (GST) gene and expressed under the control of the inducible tac promoter in Escherichia coli at 37 °C. The fusion protein with the complete preS2 region was moderately expressed (8%) while the protein with the N-terminal 26 aa was expressed at a higher level, yielding about 20% of the total cellular proteins. The GST-preS2 (aa 1–26) protein, which contains the immunodominant epitope, was produced form the soluble protein fraction of the recombinant bacteria and purified by affinity chromatography using glutathione-agarose column. The purified preS2 fusion protein showed the antigenicity of preS2, as assessed by indirect and competitive ELISAs.     

A pre-S gene chip to detect pre-S deletions in hepatitis B virus large surface antigen as a predictive marker for hepatoma risk in chronic hepatitis B virus carriers

Background  

Chronic hepatitis B virus (HBV) infection is an important cause of hepatocellular carcinoma (HCC) worldwide. The pre-S₁ and -S₂ mutant large HBV surface antigen (LHBS), in which the pre-S₁ and -S₂ regions of the LHBS gene are partially deleted, are highly associated with HBV-related HCC.     

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.     

Characteristics of pre-S2 region of hepatitis B virus

The nucleotide sequence of our cloned HBV DNA (subtype adw) has been determined. When the 165-nucleotide sequence of the pre-S2 region was compared with 7 other published sequences (subtypes adw, adr, ayw, and adyw), we found 38 nucleotide substitutions among different subtypes and 4 (adr) or 6 (adw and ayw) substitutions within the same subtype. Analysis of the predicted amino acid sequence from the known nucleotide sequences indicates that: there are 20 amino acid substitutions, the longest conserved amino acid sequence is located between amino acids 23 to 34, and the 54th amino acid is identical within the same subtype but varies in different subtypes.     

Biologically active Rep proteins of adeno-associated virus type 2 produced as fusion proteins in Escherichia coli.

Four Rep proteins are encoded by the human parvovirus adeno-associated virus type 2 (AAV). The two largest proteins, Rep68 and Rep78, have been shown in vitro to perform several activities related to AAV DNA replication. The Rep78 and Rep68 proteins are likely to be involved in the targeted integration of the AAV DNA into human chromosome 19, and the full characterization of these proteins is important for exploiting this phenomenon for the use of AAV as a vector for gene therapy. To obtain sufficient quantities for facilitating the characterization of the biochemical properties of the Rep proteins, the AAV rep open reading frame was cloned and expressed in Escherichia coli as a fusion protein with maltose-binding protein (MBP). Recombinant MBP-Rep68 and MBP-Rep78 proteins displayed the following activities reported for wild-type Rep proteins when assayed in vitro: (i) binding to the AAV inverted terminal repeat (ITR), (ii) helicase activity, (iii) site-specific (terminal resolution site) endonuclease activity, (iv) binding to a sequence within the integration locus for AAV DNA on human chromosome 19, and (v) stimulation of radiolabeling of DNA containing the AAV ITR in a cell extract. These five activities have been described for wild-type Rep produced from mammalian cell extracts. Furthermore, we recharacterized the sequence requirements for Rep binding to the ITR and found that only the A and A' regions are necessary, not the hairpin form of the ITR.     

Production of infectious hepatitis delta virus in vitro and neutralization with antibodies directed against hepatitis B virus pre-S antigens.

Hepatitis delta virus (HDV) particles were produced in Huh7 human hepatoma cells by transfection with cloned hepatitis B virus (HBV) DNA and HDV cDNA. The particles were characterized by their buoyant density, the presence of encapsidated viral RNA, and their ability to infect primary cultures of chimpanzee hepatocytes. Successful infection was evidenced by the appearance of increasing amounts of intracellular HDV RNA after exposure to particles. Infection was prevented when particles were incubated with antibodies directed against synthetic peptides specific for epitopes of the pre-S1 or pre-S2 domains of the HBV envelope proteins before exposure to hepatocytes. These data demonstrate that HDV particles produced in vitro are infectious and indicate (i) that infectious particles are coated with HBV envelope proteins that contain the pre-S1 and pre-S2 regions, (ii) that epitopes of the pre-S1 and pre-S2 domains of HBV envelope proteins are exposed at the surface of HDV particles, and (iii) that antibodies directed against those epitopes have neutralizing activity against HDV.     

Hepatitis B virus nucleocapsid/pre-S2 fusion proteins expressed in attenuated Salmonella for oral vaccination

Hybrid HBV nucleocapsid-pre-S(2) fusion proteins were stably expressed in several aromatic-dependent attenuated Salmonella typhimurium and Salmonella dublin strains. When these live recombinant bacteria were administered i.p. to BALB/c mice they induced high titer anti-hepatitis B virus core Ag (HBc) and detectable anti-pre-S2 serum antibodies. Upon oral feeding of the recombinant salmonellae to mice, the rate of seroconversion to anti-HBc was dependent on the salmonella strain used. With the best carrier strain high titer anti-HBc antibodies and lower titer anti-pre-S2 serum IgG antibodies were observed two weeks after a single oral immunization. The Ig class and IgG subclass distribution of anti-HBc antibodies after i.p. and oral immunization is consistent with the induction of functional T cell help.     

Stability of murine, chimeric and humanized antibodies against pre-S2 surface antigen of hepatitis B virus

We have constructed a humanized antibody with specificity for the pre-S2 surface antigen of hepatitis B virus (HBV) by grafting the complementarity determining regions (CDRs) of parental murine monoclonal antibody (mAb) into human anti-Sm antibody framework regions. The humanized antibody has a substitution at position 94 in a framework region of the heavy chain variable region, and exhibits the same antigen binding affinity as the parental murine monoclonal and chimeric antibodies. In order to assess the stability of these antibodies, thermal inactivation of the parental, chimeric and humanized antibodies was analyzed. Fifty percent inactivation of the chimeric and humanized antibodies was observed at 63.7 degrees C and 68.7 degrees C, respectively, compared to 55.0 degrees C for murine antibody. The humanized antibody also exhibited increased stability against denaturant. Guanidine-induced unfolding monitored by the changes in fluorescence intensity at 360 nm showed that midpoints of the transition of the chimeric and humanized antibodies were 2.47 M and 2.56 M, respectively, whereas that of the murine antibody was 1.36 M.     

Expression of hepatitis A virus recombinant proteins in Escherichia coli

On the basis of coding regions on the fragments of genes P1 and P2 of hepatitis A virus (HAV) recombinant proteins of this virus have been synthesized in the prokaryotic expressing system of E. coli, isolated and studied with the use of sera obtained from hepatitis A patients. The capacity of HAV recombinant proteins for binding with the sera of patients with hepatitis A in the acute stage has been shown with the use of immunoblotting and the indirect solid-phase enzyme immunoassay. The results obtained in this investigation are discussed in the light of the possible use of recombinant proteins for the detection of HAV markers.     

Fermentation study for the production of hepatitis B virus pre-S2 antigen by the methylotrophic yeastHansenula polymorpha

Summary 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 yeastHansenula 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 ofH. 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 that 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 recombinantH. 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.Part of these results have been presented at the Deuxième Congrès de la Société Française de Microbiologie, Strasbourg, France, September 1989.     

Study of the antigenic structure of hepatitis B virus proteins. I. Synthesis of pre-S fragments of hepatitis B virus proteins and their immunogenicity

Fragments of hepatitis B virus envelope proteins corresponding to the parts of the pre-S domain were synthesised and immobilized on the carriers with low own immunogenicity. The highest stimulation of the antibody production was observed for the antigens immobilized on microspherical carriers or gelatin modified by H-Gly-Tyr-OH. Among peptides used for immunization, pre-S fragment 134-144, conjugated with microspherical carrier, proved to be the most active.     

Expression of glycoprotein-processing enzymes in Escherichia coli as fusion proteins

A truncated Man9-mannosidase gene and a full-length -1,4-galactosyltransferase gene were isolated from human kidney and placenta cDNAs, respectively. Both genes were cloned in plasmid pMAL-c2 to produce fusions with maltose-binding protein. Fusion products were purified by affinity chromatography. Purified enzymes were assayed using pyridyl-amino labelled oligosaccharides as substrates and analysed by HPLC.     

Increased expression of hepatitis B virus surface antigen gene in Escherichia coli by IS1 insertion

Summary We selected faster growing colonies of Escherichia coli harbouring an expression plasmid for hepatitis B virus surface antigen (HBsAg) gene after mutagenesis. Among these colonies, three were found to produce an increased level of HBsAg as a consequence of alteration of the plasmid. Analysis of this plasmid showed that an insertion sequence, IS1, was inserted into a middle region of the HBsAg gene (codon for Pro 127) to generate a termination codon 20 bp downstream from the junction site between the HBsAg gene and the left end of IS1. Insertion of a chemically synthesized termination codon into the same region of the HBsAg gene also increased the expression of the HBsAg gene. These results suggest that HBsAg lacking the COOH-terminal region is produced at a high level because it does not inhibit the growth of the host.     

Expression and purification of Dengue virus type 2 envelope protein as a fusion with hepatitis B surface antigen in Pichia pastoris

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.     

Identification of polymerized-albumin receptor domain in the pre-S2 region of hepatitis B virus surface antigen M protein.

The pre-S2-coding region in the hepatitis B virus surface antigen M (P31; pre-S2 + S) protein gene was modified to identify a polymerized-albumin receptor (PAR) domain by deleting restriction fragments or performing site-directed mutagenesis. The modified M protein genes (M-P31x; x = d, e, f, h and i) were cloned into the yeast generalized-expression vector pGLD 906-1 and expressed in Saccharomyces cerevisiae under the control of yeast glyceraldehyde-3-phosphate dehydrogenase gene promoter. The PAR activities of these gene products suggested that the PAR domain is located in the hydrophilic and highly conserved domain in the pre-S2 region (around Leu12 approximately Tyr21). Antibodies specific for a pre-S2 peptide (Phe8 approximately Pro34, subtype adr), which covers the PAR domain, were purified from sera of rabbits immunized with yeast-derived M protein particles having a natural PAR domain. Immune electron microscopy showed that the purified antibodies could aggregate HBV particles. Therefore, it was speculated that the PAR domain overlapped with the dominant virus-neutralizing and virus-protecting epitopes.     

Expression and secretion of hepatitis B viral surface antigen in E. coli

Hepatitis B viral surface antigen (HBsAg) gene was subcloned into the BglII site of Bacillus licheniformis penicillinase (penP) gene of secretory vector pJP104. Expression and secretion of HBsAg protein was achieved by the E. coli CS412 carrying the plasmid pJPS2 in which the penP:HBsAg hybrid gene was under the control of two promoters, lipoprotein (lpp) and penP, spaced 450 bases apart. The secreted form of HBsAg encoded by the hybrid penP: HBsAg gene of plasmid pJPS2 was purified by immunoaffinity chromatography and found to be a 25 kilodalton protein.     

Improving expression and solubility of rice proteins produced as fusion proteins in Escherichia coli

For proteins of higher eukaryotes, such as plants, which have large genomes, recombinant protein expression and purification are often difficult. Expression levels tend to be low and the expressed proteins tend to misfold and aggregate. We tested seven different expression vectors in Escherichia coli for rapid subcloning of rice genes and for protein expression and solubility levels. Each expressed gene product has an N-terminal fusion protein and/or tag, and an engineered protease site upstream of the mature rice protein. Several different fusion proteins/tags and protease sites were tested. We found that the fusion proteins and the protease sites have significant and varying effects on expression and solubility levels. The expression vector with the most favorable characteristics is pDEST-trx. The vector, which is a modified version of the commercially available expression vector, pET-32a, contains an N-terminal thioredoxin fusion protein and a hexahistidine tag, and is adapted to the Gateway expression system. However, addition of an engineered protease site could drastically change the expression and solubility properties. We selected 135 genes corresponding to potentially interesting rice proteins, transferred the genes from cDNAs to expression vectors, and engineered in suitable protease sites N-terminal to the mature proteins. Of 135 genes, 131 (97.0%) could be expressed and 72 (53.3%) were soluble when the fusion proteins/tags were present. Thirty-eight mature-length rice proteins and domains (28.1%) are suitable for NMR solution structure studies and/or X-ray crystallography. Our expression systems are useful for the production of soluble plant proteins in E. coli to be used for structural genomics studies.