Hepatitis B virus core antigen: synthesis in Escherichia coli and application in diagnosis.

Abstract

Fragments of hepatitis B virus DNA cloned in plasmid pBR322 carrying the gene for the viral core antigen have been placed under the control of the lac promoter of Escherichia coli. Several of the new recombinants direct higher levels of synthesis of the antigen, but the degree of enhancement varies with the different structures of the plasmids and hence the mRNAs produced. The antigen in crude bacterial lysates is a satisfactory diagnostic reagent for antibodies to the core antigen in serum samples.DNA from hepatitis B virus (HBV) has been cloned and propagated in Escherichia coli as a series of fragments produced by digestion with restriction endonucleases and as entire molecules after joining to plasmid or A phage vectors (1,2,3,4). Some of the fragments of HBV DNA cloned into the Pst I restriction site in the ,B3lactamase gene carried by plasmid pBR322 direct the synthesis of HBV core antigen (HBcAg) in E. coli (1, 5), the product being readily detectable by solid-phase radioimmunoassay (6) and having the capacity to induce specific antibody synthesis in rabbits (5).To (11) and recovered by transformation as described by Lederberg and Cohen (12). Exonuclease Digestion. The ends of the hepatitis DNA fragment from pHBV139a were digested with the double-strandspecific exonuclease, BAL-31 (13), at 30'C for 4 min in a mixture of 60 jig of pHBV139a DNA digested with Pst I and 6.6 units of BAL-31 in 600 1.l of 20 mM Tris-HCl, pH 8/12 mM CaCl2/ 12 mM MgCl2/600 mM NaCl/1 mM EDTA; the reaction was stopped by extraction with phenol.Preparation of Bacterial Extracts. Cells were harvested in late logarithmic growth phase and stored at -20°C. Thawed cells (from 2 liters of culture mixture) were suspended in 6 ml of 25% (wt/vol) sucrose in 50 mM Tris-HCl, pH 8.0, and 1 ml oflysozyme (5 mg/ml) in 250 mM Tris-HCl, pH 8.0, was added. After 5 min at 0°C, 2.5 ml of0.25 M EDTA, pH 8.0, was added and, after afurther5 min at O°C, 10 ml of1% Triton X-100, 0.4% Na deoxycholate in 50 mM Tris-HCl, pH 8.0/62.5 mM EDTA was added. After 10 min at 0°C with occasional shaking, 1 ml of 1 M MgCl2 and 0.2 ml of pancreatic DNase solution (10 mg/ ml) were added; this mixture was incubated at 37°C for 1 hr and then clarified by centrifugation at 10,000 rpm for 10 min. Protein concentration was measured by the Lowry method. The clear supernatant was dispensed and stored at -20°C.Radioimmunoassay. Solid-phase radioimmunoassay was carried out on bacterial colonies (6)

Mutagenesis of murine granulocyte/macrophage-colony-stimulating factor reveals critical residues near the N terminus

“…Note that there are several instances where four or more independent deletion mutants have terminated at the same position; these positions tend to be immediately 5' of stretches of two or more deoxyguanosine residues, as has been evident in previous studies (e.g. [16]). In order to achieve even a quasirandom distribution of mutants, it was necessary to fractionate the truncated cDNA molecules into discrete size classes prior to recloning.…”

The serology of chronic hepatitis B infection revisited.

“…Recombinant HBcAg ofthe ayw subtype derived from an Escherichia coli expression vector (8) was provided by F. Schodel (Max Planck Institute, Munich). Recombinant HBeAg (an HBcAg deletion mutant lacking the carboxy-terminal 39 amino acids) of the ayw subtype derived from an E. coli expression vector (9) was provided by S. Stahl (Biogen, Geneva). The rHBeAg was maintained at pH 9.6 and expressed only HBeAg antigenicity as assessed by reactivity with antiHBe-specific mAbs, and the rHBcAg expressed only HBcAg antigenicity as determined by reactivity with anti-HBc-specific mAbs (10).…”

Separation and crystallization ofT= 3 andT= 4 icosahedral complexes of the hepatitis B virus core protein

“…The C-terminal 34 residues form the protamine domain which is required for nucleic acid packaging but not for capsid assembly (Milich et al, 1988). Full-length capsid protein and assembly domain have been expressed in Escherichia coli, where they form capsids (Pasek et al, 1979;Stahl et al, 1982;Cohen & Richmond, 1982).…”