Expression of HBcAg mutant with long internal deletion in Saccharomyces cerevisiae and observation of its self-assembly particles by atomic force microscopy (AFM).
An internally truncated C gene of adr hepatitis B virus core antigen with long internal deletion (aa81-aa116) (DeltaHBcAg with 36aa truncation) was expressed in Saccharomyces cerevisiae and the products (DeltarHBcAg) 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 indicated that the purified products (DeltarHBcAg protein) with HBeAg antigenicity mainly located at the densities of 1.23 g ml-1. Observation and analysis of the purified DeltarHBcAg products by AFM indicated that the DeltarHBcAg (core) protein produced in S. cerevisiae could self-assemble into three or more size classes of core particles which exhibited a polymorphous distribution of DeltarHBcAg (core) particles. These different size classes of core particles mainly centred on the range whose mean diameter was from 10 nm to 48 nm, especially on the position of 11 nm, 15.6 nm and the range from 27 nm to 41 nm, respectively. Furthermore, the most number of core particles mainly centred on the range whose mean diameter was from 27 nm to 41 nm. These results above indicated that the truncated internal long fragment (aa81-aa116) probably had no effect on self-assembly of the HBcAg core particles which implied the internal length fragment (aa81-aa116) was not the sole domain for self-assembly of HBcAg dimer or the truncated HBcAg protein subunit formed the fresh interactive domain with each other. These initial results above by AFM analysis were very important for further research on the self-assembly, ultrastructure, subunit interaction and core internal deletion mutant (CIDM) function of HBcAg core particles.
Chen
Heng
H
School of Life Sciences, Shanghai University, Shanghai 200444, PR China. hengc@ualberta.ca
Liu
ShuPeng
S
Chen
Li
L
Huang
JieHuan
J
Xiang
ShaoMing
S
D000595:Amino Acid Sequence; D001483:Base Sequence; D002586:Cesium; D002712:Chlorides; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D006512:Hepatitis B Core Antigens / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D018625:Microscopy, Atomic Force; D008969:Molecular Sequence Data; D012441:Saccharomyces cerevisiae; D017384:Sequence Deletion; D014461:Ultracentrifugation
D002712:Chlorides; D006512:Hepatitis B Core Antigens; D002586:Cesium; C028019:cesium chloride