Assembly of human papillomavirus type-16 virus-like particles: multifactorial study of assembly and competing aggregation.
Pentameric capsomeres of human papillomavirus capsid protein L1 expressed in Escherichia coli self-assemble into virus-like particles (VLPs) in vitro. A multifactorial experimental design was used to explore a wide range of solution conditions to optimize the assembly process. The degree of assembly was measured using an enzyme-linked immunosorbent assay, and a high-throughput turbidity assay was developed to monitor competing aggregation. The presence of zinc ions in the assembly buffer greatly increased the incidence of aggregation and had to be excluded from the experiment for meaningful analysis. Assembly of VLPs was optimal at a pH of about 6.5, calcium and sodium ions had no measurable effect, and dithiothreitol and glutathione inhibited assembly. Tryptophan fluorescence spectroscopy demonstrated that an increase in urea concentration reduced the rate of VLP formation but had no effect on the final concentration of assembled VLPs. This study demonstrates the use of the hanging-drop vapor-diffusion crystallization method to screen for conditions that promote aggregation and the use of tryptophan fluorescence spectroscopy for real-time monitoring of the assembly process.
Hanslip
Simon J
SJ
Department of Chemical Engineering, University of Cambridge, Pembroke Street, Cambridge, CB2 3RA, UK.
Zaccai
Nathan R
NR
Middelberg
Anton P J
AP
Falconer
Robert J
RJ
D002413:Cations, Divalent / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay; D005978:Glutathione / Q000378:metabolism; D052162:Human papillomavirus 16 / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D008854:Microscopy, Electron; D008958:Models, Molecular; D008968:Molecular Conformation; D010084:Oxidation-Reduction; D014364:Tryptophan / Q000737:chemistry; D014508:Urea; D014771:Virion / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure
D002413:Cations, Divalent; D014364:Tryptophan; D014508:Urea; D005978:Glutathione