Journal of Electron Microscopy Advance Access originally published online on October 31, 2007
Journal of Electron Microscopy 2007 56(6):235-242; doi:10.1093/jmicro/dfm028
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Expression and purification of the recombinant HBcAg core particles derived from methyltrophic Pichia pastoris, and TEM and AFM of the core particles and their natural aggregates
1 School of Life Science, Shanghai University, Shanghai 200444, P.R. China
2 College of Life Science and Biotechnology, Shanghai Jiaotong University, Shanghai 200030, P.R. China
* To whom correspondence should be addressed. E-mail: hengc{at}ualberta.ca
The recombinant hepatitis B virus core antigen (rHBcAg) core particles derived from Pichia pastoris were purified from a crude lysate of the yeast by three steps: Sephrose CL-4B chromatography, sucrose step-gradient ultracentrifugation and CsCl-isopycnic ultracentrifugation. Results of ELISA test and density analysis of CsCl-isopycnic ultracentrifugation indicate that the purified rHBcAg particles with HBcAg antigenicity mainly locate at the densities of 1.2576 and 1.3013 g·mL–1, respectively. After purification, a portion of purified sample of rHBcAg particles was immediately subjected to detection using transmission electron microscopy (TEM) and atomic force microscopy (AFM), the remainder were kept in –20°C for 1 month or longer. After 30 days, the sample of rHBcAg particles previously frozen was imaged by TEM and AFM. The detection results indicate that the stored rHBcAg particles aggregated into a string of beads. The above results suggested that the rHBcAg particles expressed and self-assembled in P. pastoris, which were stored at –20°C, can gradually and naturally aggregate with storage time.
Keywords aggregation, atomic force microscopy (AFM), rHBcAg core particle, transmission electron microscopy (TEM)
Abbreviations: AFM, atomic force microscopy; • EM, electron microscopy; • ELISA, enzyme-linked immunosorbent assay; • HBV, hepatitis B virus; • HBcAg, HBV core antigen; • P. pastoris, Pichia pastoris; • rAAV-2, recombinant adeno-associated virus type 2; • rHBcAg, recombinant hepatitis B core antigen; • rHBsAg, recombinant hepatitis B surface antigen; • 2D, two-dimensional; • 3D, three-dimensional; • TEM, transmission electron microscopy.;
Received 23 February 2007, accepted 18 September 2007
The originally published version of this article was incorrect. In Fig. 9, the graphs were displayed in µm rather than nm. The publisher apologizes for this error.