title: Development of an IP-Free Biotechnology Platform for Constitutive Production of HPV16 L1 Capsid Protein Using the Pichia pastoris PGK1 Promoter.
abstract: The human papillomavirus (HPV) L1 major capsid protein, which forms the basis of the currently available vaccines against cervical cancer, self-assembles into virus-like particles (VLPs) when expressed heterologously. We report the development of a biotechnology platform for HPV16 L1 protein expression based on the constitutive PGK1 promoter (PPGK1) from the methylotrophic yeast Pichia pastoris. The L1 gene was cloned under regulation of PPGK1 into pPGKΔ3 expression vector to achieve intracellular expression. In parallel, secretion of the L1 protein was obtained through the use of an alternative vector called pPGKΔ3α, in which a codon optimized α-factor signal sequence was inserted. We devised a work-flow based on the detection of the L1 protein by dot blot, colony blot, and western blot to classify the positive clones. Finally, intracellular HPV VLPs assembly was demonstrated for the first time in yeast cells. This study opens up perspectives for the establishment of an innovative platform for the production of HPV VLPs or other viral antigens for vaccination purposes, based on constitutive expression in P. pastoris.
authors.affiliation: Laboratorio de Estudos Moleculares e Terapia Experimental, Departamento de Genética, Universidade Federal de Pernambuco, 50670-901 Recife, PE, Brazil ; Departamento de Microbiologia, Centro de Pesquisas Aggeu Magalhaes, 50740-465 Recife, PE, Brazil.
authors.forename: F C
authors.initials: FC
authors.lastname: Mariz
authors.affiliation: Laboratorio de Estudos Moleculares e Terapia Experimental, Departamento de Genética, Universidade Federal de Pernambuco, 50670-901 Recife, PE, Brazil.
authors.forename: E C
authors.initials: EC
authors.lastname: Coimbra
authors.affiliation: Laboratorio de Estudos Moleculares e Terapia Experimental, Departamento de Genética, Universidade Federal de Pernambuco, 50670-901 Recife, PE, Brazil.
authors.forename: A L S
authors.initials: AL
authors.lastname: Jesus
authors.affiliation: Departamento de Microbiologia, Centro de Pesquisas Aggeu Magalhaes, 50740-465 Recife, PE, Brazil.
authors.forename: L M
authors.initials: LM
authors.lastname: Nascimento
authors.affiliation: Laboratorio de Biologia Molecular, Departamento de Biologia Celular, Universidade de Brasília, 70910-900 Brasília, DF, Brazil.
authors.forename: F A G
authors.initials: FA
authors.lastname: Torres
authors.affiliation: Laboratorio de Estudos Moleculares e Terapia Experimental, Departamento de Genética, Universidade Federal de Pernambuco, 50670-901 Recife, PE, Brazil.
authors.forename: A C
authors.initials: AC
authors.lastname: Freitas
mesh_terms: D001709:Biotechnology; D036022:Capsid Proteins / Q000737:chemistry / Q000276:immunology*; D005260:Female; D015967:Gene Expression Regulation, Viral; D052162:Human papillomavirus 16 / Q000276:immunology / Q000472:pathogenicity; D006801:Humans; D009856:Oncogene Proteins, Viral / Q000737:chemistry / Q000276:immunology*; D010843:Pichia; D011401:Promoter Regions, Genetic; D002583:Uterine Cervical Neoplasms / Q000517:prevention & control* / Q000821:virology; D058425:Vaccines, Virus-Like Particle
chemical_list: D036022:Capsid Proteins; C114580:HPV L1 protein, Human papillomavirus; D009856:Oncogene Proteins, Viral; D058425:Vaccines, Virus-Like Particle