title: Expression and characterization of HPV-16 L1 capsid protein in Pichia pastoris.
abstract: Human papillomaviruses (HPVs) are responsible for the most common human sexually transmitted viral infections. Infection with high-risk HPVs, particularly HPV16, is associated with the development of cervical cancer. The papillomavirus L1 major capsid protein, the basis of the currently marketed vaccines, self-assembles into virus-like particles (VLPs). Here, we describe the expression, purification and characterization of recombinant HPV16 L1 produced by a methylotrophic yeast. A codon-optimized HPV16 L1 gene was cloned into a non-integrative expression vector under the regulation of a methanol-inducible promoter and used to transform competent Pichia pastoris cells. Purification of L1 protein from yeast extracts was performed using heparin-sepharose chromatography, followed by a disassembly/reassembly step. VLPs could be assembled from the purified L1 protein, as demonstrated by electron microscopy. The display of conformational epitopes on the VLPs surface was confirmed by hemagglutination and hemagglutination inhibition assays and by immuno-electron microscopy. This study has implications for the development of an alternative platform for the production of a papillomavirus vaccine that could be provided by public health programs, especially in resource-poor areas, where there is a great demand for low-cost vaccines.
authors.affiliation: Centro de Biotecnologia, Instituto Butantan, Av. Vital Brasil, Sao Paulo, SP, Brazil.
authors.forename: Silvia Boschi
authors.initials: SB
authors.lastname: Bazan
authors.forename: Agtha
authors.initials: A
authors.lastname: de Alencar Muniz Chaves
authors.forename: Karina Araújo
authors.initials: KA
authors.lastname: Aires
authors.forename: Aurora Marques
authors.initials: AM
authors.lastname: Cianciarullo
authors.forename: Robert L
authors.initials: RL
authors.lastname: Garcea
authors.forename: Paulo Lee
authors.initials: PL
authors.lastname: Ho
mesh_terms: D015153:Blotting, Western; D036022:Capsid Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002472:Cell Transformation, Viral / Q000502:physiology; D004591:Electrophoresis, Polyacrylamide Gel; D015967:Gene Expression Regulation, Viral; D006385:Hemagglutination Inhibition Tests; D052162:Human papillomavirus 16 / Q000378:metabolism* / Q000648:ultrastructure; D046529:Microscopy, Electron, Transmission; D009856:Oncogene Proteins, Viral / Q000302:isolation & purification / Q000378:metabolism*; D030361:Papillomavirus Infections / Q000378:metabolism; D010843:Pichia / Q000378:metabolism / Q000821:virology*
chemical_list: D036022:Capsid Proteins; D009856:Oncogene Proteins, Viral; C063317:L1 protein, Human papillomavirus type 16