The hepatitis B virus (HBV) envelope (env) protein is composed of three regions: the 108- or 119-residue pre-Si region involved in the direct interaction with hepatocytes. the 55-residue pre-SZ region associated with the polymerized albumin-mediated interaction, and the major 226-residue S protein region. Thus, to improve the immunogenic potency of conventional HE vaccines, development of a new vaccine containing the entire pre-Si region in addition to pre-S2 and S is desired. We previously reported the efficient production of the HBV env L (pre-S1 + pre-S2 + S) protein in the recombinant yeast cells [J Biol Chem 267 (1992) 1953]. In this study, the HBV env L protein produced as nano-particles in yeast has been purified and characterized. By equilibrium sedimentation. an average molecular weight of L particle was estimated to be approximately 6.3 x 10(6), indicating that about 110 molecules of L proteins are assembled into an L particle. By atomic force microscopy in a moist atmosphere. the L particles were observed as large spherical particles with a diameter of 50-500 nm. The L particles were stable on short-time heating at a high temperature and long-time storage at a low temperature but rather unstable on repeated freezing and thawing and treatment with dithiothreitol. When immunized in mice. L particles elicited efficiently and simultaneously the anti-g, anti-pre-S2, and anti-pre-Si antibodies. The ED50 values in mice for the anti-S and anti-pre-S2 antibodies were similar to those elicited by the M (pre-S2 + S) particles. Furthermore. the anti-pre-Si rabbit antibodies were found to recognize various segments of the pre-Si region, including the pre-Si (21-47) segment. These results show the high ability of L particles to induce all antibodies against HBV env proteins, hence promising the future application of L particles for the next generation HE vaccine. (C) 2001 Elsevier Science Ltd. All rights reserved.