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.     

Expression of a DNA methylation (dam) gene inEscherichia coli K-12

Plasmid pMQ3, carrying thedam gene ofEscherichia coli on a 6.1 Kb fragment, shows a tenfold increase in relative DNA adenine methylase activity, while plasmid pdam118, with a 1.14 Kbdam insert, shows only a twofold increase, although both plasmids were derived from plasmid pLC13–42. Since a copy number effect did not seem to be the cause of this difference, we have subcloned pMQ3 in order to determine whether the additional chromosomal DNA present in this plasmid is responsible for the enhancement of methylase activity. We show that the 346 base pairs upstream ofdam contain sequences necessary for expression. DNA sequence analysis has revealed that in pdam118 only the 118 bases 5-prime to thedam gene are present in other constructs and that the additional upstream material is pBR322 DNA. This shows that pdam118 carries a DNA duplication.     

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.     

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.     

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 an endoglucanase gene fromClostridium cellulolyticum inEscherichia coli

Summary A gene coding for an endoglucanase from the anaerobic cellulolytic bacteriumClostridium cellulolyticum has been cloned by direct selection inEscherichia coli, using the carboxymethyl cellulose-Congo Red assay. The cloned gene has been subcloned in the two possible orientations in pUC plasmids. One of the two resulting constructs exhibited a higher level of expression, which was associated with a high level of plasmid instability. The enzyme synthesized inE. coli from the cloned gene has been characterized by two procedures, maxicells and gel filtration chromatography, as a polypeptide of approximately 40 kilodaltons.     

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.     

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.     

Expression of hepatitis B virus surface and core antigens: influences of pre-S and precore sequences.

Amphotropic retroviral expression systems were used to synthesize hepatitis B virus surface antigen (HBsAg) and core antigen. The vectors permitted establishment of cell lines which expressed antigen from either the retroviral long terminal repeat or the mouse metallothionein-I promoter. HBsAgs were synthesized containing no pre-S sequences, pre-S(2) sequences alone, or pre-S(1) plus pre-S(2) sequences. Inclusion of pre-S(2) sequences did not affect the secretion or density of HBsAg particles but did reduce their mass by approximately 30%. Addition of pre-S(1) sequences almost completely abolished secretion of HBsAg and resulted in its localization in an aqueous-nonextractable pre- or early-Golgi cellular compartment. HBsAg was localized to the cytoplasm of the cell. This localization was unaffected by the presence of pre-S sequences in the antigen. Cell lines synthesizing hepatitis B antigens from core DNA fragments, containing or not containing precore sequences, secreted hepatitis B e antigen. However, the absence of precore DNA sequences resulted in additional synthesis of hepatitis core antigen, which was predominantly nuclear in localization.     

Expression of theunc genes inEscherichia coli

Theunc (or atp) operon ofEscherichia coli comprises eight genes encoding the known subunits of the proton-translocating ATP synthase (H⁺-ATPase) plus a ninth gene (uncI) of unknown function. The subunit stoichiometry of the H⁺-ATPase ( ₃₃₁₁₁a₁b₂c_10–15) requires that the respectiveunc genes be expressed at different rates. This review discusses the experimental methods applied to determining how differential synthesis is achieved, and evaluates the results obtained. It has been found that the primary level of control is translational initiation. The translational efficiencies of theunc genes are determined by primary and secondary mRNA structures within their respective translational initiation regions. The respective rates of translation are matched to the subunit requirements of H⁺-ATPase assembly. Finally, points of uncertainty remain and experimental strategies which will be important in future work are discussed.     

Mapping of the hepatitis B virus pre-S1 domain involved in receptor recognition

Hepatitis B virus (HBV) and woolly monkey hepatitis B virus (WMHBV) are primate hepadnaviruses that display restricted tissue and host tropisms. Hepatitis D virus (HDV) particles pseudotyped with HBV and WMHBV envelopes (HBV-HDV and WM-HDV) preferentially infect human and spider monkey hepatocytes, respectively, thereby confirming host range bias in vitro. The analysis of chimeric HBV and WMHBV large (L) envelope proteins suggests that the pre-S1 domain may comprise two regions that affect infectivity: one within the amino-terminal 40 amino acids of pre-S1 and one downstream of this region. In the present study, we further characterized the role of the amino terminus of pre-S1 in infectivity by examining the ability of synthetic peptides to competitively block HDV infection of primary human and spider monkey hepatocytes. A synthetic peptide representing the first 45 residues of the pre-S1 domain of the HBV L protein blocked infectivity of HBV-HDV and WM-HDV, with a requirement for myristylation of the amino terminal residue. Competition studies with truncated peptides suggested that pre-S1 residues 5 to 20 represent the minimal domain for inhibition of HDV infection and, thus, presumably represent the residues involved in virus-host receptor interaction. Recombinant pre-S1 proteins expressed in insect cells blocked infection with HBV-HDV and WM-HDV at a concentration of 1 nanomolar. The ability of short pre-S1 peptides to efficiently inhibit HDV infection suggests that they represent suitable ligands for identification of the HBV receptor and that a pre-S1 mimetic may represent a rational therapy for the treatment of HBV infection.     

Expression ofChlamydia psittaci-encoded polypeptides inEscherichia coli

DNA fromChlamydia psittaci was partially digested with Sau 3A and cloned into the lambda bacteriophage derivative vector Charon 30. From this bank about 30 of 1500 recombinants reacted with rabbit anti-C. psittaci serum. Fourteen of these clones expressed antigens varying between 15 and 76 kilodaltons (kd) as revealed by SDS-PAGE and immunoblotting. Two recombinants, expressing 27-kd and 72-kd+76-kd antigens, respectively, were further analyzed by immunoblotting with rabbit antiserum and sera from humans with different chlamydial infections. Partial restriction endonuclease cleavage of these clones showed 10 and 13 kilobases inserts, respectively.     

Expression ofMycobacterium tuberculosis genes inEscherichia coli

TwoEscherichia coli clones expressingMycobacterium tuberculosis antigens were isolated from a gene-bank in the plasmid vector pBR 325. ‘Western blot’ analysis revealed the presence of a unique protein band of molecular weight 68,000 and 38,000, respectively in cellextracts from each clone. The 68,000 dalton antigen was found to be expressed onEscherichia coli outer surface. Plasmid DNA from a third clone could confer leucine independence on two differentleu B mutants ofEscherichia coli but not on mutants in otherleu genes, pointing to the possibility ofgenetic complementation. Thus,Mycobacterium tuberculosis DNA is capable of expression inEscherichia coli.     

Characterization of purified hepatitis B surface antigen containing pre-S(2) epitopes expressed in Saccharomyces cerevisiae

The cloning and expression of the hepatitis B middle-protein surface antigen gene in the yeast Saccharomyces cerevisiae is described. A generalized expression vector carrying the yeast glyceraldehyde-3-phosphate dehydrogenase gene promoter was used. Expressed material, in the form of supramolecular particles, was purified and characterized. Severe proteolysis within the pre-S(2) region was observed for material expressed in a wild-type yeast host. This proteolysis was substantially reduced by utilization of a protease-deficient host. Immunoblotting of sodium dodecyl sulfate-polyacrylamide gels with several antibodies of differing specificity was performed to characterize the various protein species present. All species were analyzed by N-terminal sequencing after electroelution from gels. Carbohydrate staining of gels and glycosidase treatments of the purified antigen material indicated that full-length antigen was present in both glycosylated and unglycosylated forms. Glycosylation appeared to be of both asparagine-linked and threonine/serine-linked types. Site-directed mutagenesis was used to convert two arginine residues in the pre-S(2) region of the antigen to glutamine residues. The changes abolished reactivity with one polyclonal and two monoclonal antibodies specific for epitopes within the pre-S(2) region.     

Immune response to the pre-S(1) region of the hepatitis B surface antigen (HBsAg): a pre-S(1)-specific T cell response can bypass nonresponsiveness to the pre-S(2) and S regions of HBsAg

Hepatitis B surface antigen (HBsAg) particles are composed of a major polypeptide, p25, and additional polypeptides of higher m.w., namely p33 and p39, are variably present. All three polypeptides share the 226 amino acid residues of the S region: p33 consists of the p25 sequence plus an NH2-terminal 55 residues (pre-S(2], and p39 consists of the p33 sequence plus an NH2-terminal 108-119 residues (pre-S(1). In previous studies we demonstrated the influence of two Ir genes on the humoral and cellular immune responses to the S region and identified nonresponder phenotypes (H-2f,s). Subsequent studies showed that the immune response to the pre-S(2) region was regulated by H-2-linked genes independently of the S region response, such that immunization of S region nonresponder, pre-(S2) region responder mice (H-2s) with HBsAg/p33 circumvented nonresponse to the S region. In the present study, we have extended this analysis to the pre-S(1) region of HBsAg, with the following results: 1) and pre-S(1) region is immunogenic at the T and B cell levels; 2) anti-pre-S(1) specific antibody production is regulated by H-2-linked genes and can be independent of anti-S and anti-pre-S(2) antibody production; 3) immunization of H-2f strains with HBsAg/p39 particles containing the pre-S(1) region can bypass nonresponsiveness to the S and pre-S(2) regions in terms of antibody production; 4) two synthetic peptides, p32-53 and p94-117, define murine and human antibody binding sites on the pre-S(1) region, and p1-21 and p12-32 define additional human antibody binding sites; 5) pre-S(1)-specific T cells can be elicited in S and pre-S(2) region nonresponder mice (H-2f) and provide functional T cell help for S-pre-S(2)-, and pre-S(1)-specific antibody production; and 6) a T cell recognition site in the pre-S(1) region, p12-32 was identified. These results are relevant to HBV vaccine development, and possibly to viral clearance mechanisms, since the higher m.w. polypeptides are preferentially expressed on intact virions.     

Expression of streptomycete cholesterol oxidase inEscherichia coli

Summary A streptomycete gene coding for extracellular cholesterol oxidase (choA) was subcloned and expressed inEscherichia coli. The pUCO series recombinants were obtained by inserting thechoA gene into the uniqueKpnI site of pUC19 vector. Expression was observed with pUCO192A and pUCO193 constructs in which the cloned gene(s) were aligned with the upstreamlacZ promoter. Isopropyl -d-thioglucopyranoside (IPTG) enhanced this expression up to 2.5-fold. Specific Cho activity in the cell extracts of the stable pUCO193 transformant were 0.004 U and 0.007 U per mg protein without and with IPTG induction, respectively. Cho activity was detected in the spent medium of this culture, suggesting possible secretion of the enzyme.     

The N-terminal (pre-S2) domain of a hepatitis B virus surface glycoprotein is translocated across membranes by downstream signal sequences.

The coding region for the hepatitis B virus surface antigens contains three in-phase ATG codons which direct the synthesis of three related polypeptides. The 24-kilodalton major surface (or S) glycoprotein is initiated at the most distal ATG and is a transmembrane protein whose translocation across the bilayer is mediated by at least two uncleaved signal sequences. The product of the next upstream ATG is the 31-kilodalton pre-S2 protein, which contains 55 additional amino acids attached to the N terminus of the S protein. This pre-S2-specific domain is translocated into the endoplasmic reticulum. Using a coupled in vitro translation-translocation system, we showed that (i) the pre-S2 domain itself lacks functional signal sequence activity, (ii) its translocation across the endoplasmic reticulum membrane is mediated by downstream signals within the S domain, and (iii) the N-terminal signal sequence of the S protein can translocate upstream protein domains in the absence of other signals. The hepatitis B virus pre-S2 protein is an example of a natural protein which displays upstream domain translocation, a phenomenon whose existence was originally inferred from the behavior of synthetic fusion proteins in vitro.