Virus-like particles (VLPs) of the recombinant hepatitis B virus (HBV) core protein (HBc) are routinely used in HBV diagnostics worldwide and are of potential interest as carriers of foreign peptides (e g immunological epitopes and targeting addresses and/or as vessels for packaged diagnostic and therapeutic nanomaterials) Despite numerous reports exploiting different expression systems a rapid and comprehensive large-scale methodology for purification of HBc VLPs from yeast is still lacking Here we present a convenient protocol for highly efficient production and rapid purification of endotoxin-free ayw subtype HBc VLPs from the methylotrophic yeast Pichia pastoris The HBc gene expression cassette along with the geneticin resistance gene was transferred to the P pastoris genome via homologous recombination A producer clone was selected among 2000 transformants for the optimal synthesis of the target protein Fermentation conditions were established ensuring biomass accumulation of 163 g/L A simple combination of pH/heat and salt treatment followed by a single anion-exchange chromatography step resulted in a more than 90% pure preparation of HBc VLPs with a yield of about 3 0 mg per 1 g of wet cells Purification is performed within a day and may be easily scaled up if necessary The quality of HBc VLPs was verified by electron microscopy Mass spectrometry analysis and direct polyacrylamide gel staining revealed phosphorylation of HBc at at least two sites To our knowledge this is the first report of HBc phosphorylation in yeast (C) 2010 Elsevier Inc All rights reserved