Visualization of discrete L1 oligomers in human papillomavirus 16 virus-like particles by gel electrophoresis with Coomassie staining.
The recombinant major capsid protein (L1) of human papillomavirus (HPV) can self-assemble into virus-like particles (VLPs) with 360 L1 molecules per VLP. These tightly associated L1 oligomers in the assembled VLPs were disrupted in a pH-, denaturant-, time-, and temperature-dependent fashion. With non-reducing Laemmli-type SDS-PAGE, primarily the monomeric L1 protein ( approximately 55 kDa) is observed when analyzing VLP preparations. When the pH was lowered to pH 7.0 in NuPAGE system and the gel temperature during electrophoresis was maintained at a lower temperature ( approximately 7 degrees C), a ladder of protein bands in approximately 55 kDa increments were detected above the monomeric p55 band. These discrete bands visualized as a ladder are likely the disulfide-linked L1 oligomers. In addition to the gel running conditions, an increase in pH, temperature, or SDS concentration during sample treatment was also shown to significantly reduce the amount of detectable oligomers, further corroborating the labile nature of these oligomers. Altogether, the results also implicate the redox-responsive nature of the HPV capsid comprising of >95% L1 protein. Molecular basis of the facile disulfide bond inter-change is discussed. This electrophoretic technique for trapping the disulfide-linked oligomers may be employed to detect the oligomeric status of other protein aggregates or assembled particles.
Zhao
Qinjian
Q
Department of Bioprocess & Bioanalytical Research, Merck Research Laboratories, West Point, PA 19486, USA. qinjian_zhao@merck.com
Guo
Helen H
HH
Wang
Yang
Y
Washabaugh
Michael W
MW
Sitrin
Robert D
RD
D036022:Capsid Proteins; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics; D004587:Electrophoresis, Agar Gel; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009856:Oncogene Proteins, Viral; D027383:Papillomaviridae / Q000737:chemistry* / Q000378:metabolism; D013194:Staining and Labeling; D019065:Virus Assembly
D036022:Capsid Proteins; D004279:DNA, Viral; D009856:Oncogene Proteins, Viral; C063317:L1 protein, Human papillomavirus type 16