Disassembly and reassembly of yeast-derived recombinant human papillomavirus virus-like particles (HPV VLPs).
The human papillomavirus (HPV) virus-like particles (VLPs) produced by recombinant expression systems are promising candidate vaccine antigens for prevention of cervical cancers as well as genital warts. However, expression of HPV type 6, 11, and 16 L1 proteins in Saccharomyces cerevisiae yielded irregularly shaped, broadly distributed VLPs smaller in size (30-50 nm) than expected (60 nm). In this study, we demonstrate that these HPV VLPs can be disassembled into the constituent capsomers (L1 pentamers) by incubation at low ionic strength and elevated pH in the presence of relatively low concentration of reducing agents. Following the removal of reducing agents, lowering of pH and increasing of ionic strength, the capsomers spontaneously reassembled into homogenous, 60-nm VLPs characterized by significantly enhanced structural stability and improved immunogenicity. In order to achieve quantitative recovery of HPV VLPs, the disassembly/reassembly process was further optimized by use of high ionic strength (>0.5 M sodium chloride) to prevent aggregation of VLPs. The reassembled VLPs possess an architectural structure very similar to that of the natural HPV virus particles. This development illustrates how the natural, in vivo mechanisms facilitating cell entry and virus replication can be utilized to achieve an optimal, in vitro assembly state of yeast-expressed HPV VLPs.
Mach
Henryk
H
Merck Research Laboratories, West Point, Pennsylvania 19486, USA.
Volkin
David B
DB
Troutman
Robert D
RD
Wang
Bei
B
Luo
Zheng
Z
Jansen
Kathrin U
KU
Shi
Li
L
D036022:Capsid Proteins / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D046529:Microscopy, Electron, Transmission; D027383:Papillomaviridae; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000648:ultrastructure; D012441:Saccharomyces cerevisiae / Q000235:genetics; D014461:Ultracentrifugation; D014771:Virion
D036022:Capsid Proteins; D011994:Recombinant Proteins