Multiple chemical forms of hepatitis B surface antigen produced in yeast.

Abstract

Hepatitis B surface antigen (HBsAg) has been extracted from yeast cells that produce HBsAg. These cells contain the gene for surface antigen carried on a plasmid that replicates in the cells. Analysis of the yeast-derived HBsAg by sucrose gradient centrifugation and by polyacrylamide gel electrophoresis shows that the antigen that is initially released from yeast cells is a high molecular weight aggregate of the fundamental Mr 25,000 subunit. Unlike HBsAg derived from human plasma, the yeast antigen is held together by noncovalent interactions and can be dissociated in 2% NaDodSO4 without the use of reducing agents. During in vitro purification of the yeast antigen, some disulfide bonds form spontaneously between the antigen subunits, resulting in a particle composed of a mixture of monomers and disulfide-bonded dimers. Treatment with 3 M thiocyanate converts the 20-nm particles into a fully disulfide-bonded form that is not disrupted in NaDodSO4 unless a reducing agent is added. This disulfidebonded particle resembles the naturally occurring, plasmaderived surface antigen particle, and the in vitro formed particle has been used to prepare a vaccine for humans against hepatitis B virus infection.

Maturation of Recombinant Hepatitis B Virus Surface Antigen Particles

“…rHBsAg was overexpressed in yeast (Saccharomyces cerevisae) and purified according to previously published procedures. 6 The purified rHBsAg was used at concentrations in the range of 40 to 400 µg/mL and was stored or diluted in phosphate-buffered saline (PBS). Protein concentrations were determined by a Lowry method with commercially available reagents (Pierce).…”

“…[7][8][9] rHBsAg expressed in yeast or mammalian cells also spontaneously forms disulfide-linked dimers and higher-order oligomers during expression, and the cross-linking continues during downstream purification and storage. 6 Studies on the conformational flexibility and dynamics of this lipidassociated oligomeric protein complex and its morphological characterization throughout the maturation process have not been reported. In order to quantitatively analyze these changes, three different samples with different degrees of maturation/aging (shown in Fig.…”

“…Intra-and intermolecular disulfide bond formation occurs in the endoplasmic reticulum and pre-Golgi compartment, 5 and additional disulfide bonds are formed after cell breakage during purification and storage. 6 The importance of the correct intra-and intermolecular disulfide bonds is well documented for the immunochemical and biological function of the HBsAg subviral particles: reduction of these disulfide bonds greatly decreases or abolishes both the antigenic and immunogenic properties of this protein complex. [7][8][9] rHBsAg expressed in yeast or mammalian cells also spontaneously forms disulfide-linked dimers and higher-order oligomers during expression, and the cross-linking continues during downstream purification and storage.…”

Toward the development of monoclonal antibody-based assays to probe virion-like epitopes in hepatitis B vaccine antigen

“…[17][18][19] The top of each S-protein tetramer forms a protrusion, where it displays the critical clinically relevant epitopes. 5 The MHR projected out of the lipid layer surface is a tetramer of S-protein monomers, thus containing a total of 32 cysteine residues per protrusion.…”

“…12,[28][29][30] In early years of vaccine development, HBsAg VLP characterization was performed mainly with SDS-PAGE gel to show the degree of cross-linking, lacking information on 3-dimensional features of key epitopes. 19 Now with different anti-HBsAg mAbs against various epitopes available, the proposed assays with mAbs of choice can be performed on process intermediates or final products, yielding quantitative data and orthogonal information on antigen content ("mass ELISA") or integrity of clinically relevant epitopes (e.g., RF-1, or A1.2-like mAb binding activity, a surrogate marker for vaccine clinical efficacy).…”