Expression of human papillomavirus type 16 L1 protein in Escherichia coli: denaturation, renaturation, and self-assembly of virus-like particles in vitro.
Major capsid protein L1 of HPV16 was produced in a fused form in Escherichia coli using an inducible expression system. The protein formed insoluble aggregations (inclusion bodies) and the yield was more than 10% of total cell proteins. The inclusion bodies were isolated and solubilised with 8 M urea and the L1 proteins were purified by chromatographic separation. Following removal of the urea by gradual dialysis, the denatured L1 proteins spontaneously renatured and subsequently assembled into polymorphologic aggregations in vitro. Electron microscopy showed that the assembled material included structures resembling native empty capsids as well as incompletely formed capsids. After separation from the pool of polymorphologic structures by sucrose gradient sedimentation, the correctly formed virus-like particles (VLE. coliPs) were recognised by a HPV16 type-specific, conformational-dependent monoclonal antibody in an ELISA. This system offers not only a model for investigation of the intrinsic interactions that occur during L1 assembly, but also a potential route for convenient manufacture of highly purified VLP vaccines.
Zhang
W
W
Department of Pathology, University of Cambridge, United Kingdom.
Carmichael
J
J
Ferguson
J
J
Inglis
S
S
Ashrafian
H
H
Stanley
M
M
D036022:Capsid Proteins; D002460:Cell Line; D004926:Escherichia coli / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D009856:Oncogene Proteins, Viral / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism / Q000502:physiology*; D027383:Papillomaviridae / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D011489:Protein Denaturation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism / Q000502:physiology*; D014771:Virion; D019065:Virus Assembly
D036022:Capsid Proteins; D009856:Oncogene Proteins, Viral; D011993:Recombinant Fusion Proteins; C063317:L1 protein, Human papillomavirus type 16