Purification of the recombinant hepatitis B virus core antigen (rHBcAg) produced in the yeast Saccharomyces cerevisiae and comparative observation of its particles by transmission electron microscopy (TEM) and atomic force microscopy (AFM).
Hepatitis B virus core antigen (HBcAg) gene (C gene) was expressed in Saccharomyces cerevisiae and the products (rHBcAg or core particles) were purified from a crude lysate of the yeast by three steps: Sephrose CL-4B chromatography, Sucrose step-gradient ultracentrifugation and CsCl-isopycnic ultracentrifugation. It has been observed that HBcAg was synthesized in yeast cells as a particle consisting of polypeptides with a molecular weight of 21.5 kDa (p21.5). Results of ELISA test and density analysis of CsCl-isopycnic ultracentrifugation indicated that the purified products (rHBcAg particles) with HBcAg antigenicity mainly located at the densities of 1.27 and 1.40 g ml(-1), respectively. Observation and analysis of the purified rHBcAg products by TEM indicated that rHBcAg peptides could mainly self-assemble into two size classes of core particles. The larger particles were approximately 30.1 nm and the smaller were approximately 21.5 nm in mean diameter. Further observation and analysis of the same rHBcAg (core) particles by AFM also indicated that rHBcAg (core) particles were similar to the native HBcAg (core) particles from infected human hepatocytes and mainly composed of two size classes of partides core. The larger particles were approximately 31.3 nm and the smaller were approximately 22.5 nm in mean diameter which was similar to the results obtained by TEM. All results from both TEM and AFM suggested that core particles (capsids) produced in S. cerevisiae possessed dimorphism.
College of Life Science adn Biotechnology, Shanghai Jiaotong University, Shanghai, China. dr.chenheng@163.com
Heng
H
Chen
Jun Hong
JH
Lü
Wan Qi
WQ
Liang
Ya Hong
YH
Huang
Wei Jie
WJ
Zhang
Da Bing
DB
Zhang
D005814:Genes, Viral; D006512:Hepatitis B Core Antigens / Q000235:genetics / Q000302:isolation & purification* / Q000648:ultrastructure*; D006515:Hepatitis B virus / Q000235:genetics / Q000276:immunology; D018625:Microscopy, Atomic Force; D008854:Microscopy, Electron; D010316:Particle Size; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000648:ultrastructure; D012441:Saccharomyces cerevisiae / Q000235:genetics
D006512:Hepatitis B Core Antigens; D011994:Recombinant Proteins