Production of L1 protein from different types of HPV in Pichia pastoris using an integrative vector

Human papillomavirus (HPV) infection is the most common sexually transmitted disease in the world and is related to the etiology of cervical cancer. The most common high-risk HPV types are 16 and 18; however, the second most prevalent type in the Midwestern region of Brazil is HPV-33. New vaccine strategies against HPV have shown that virus-like particles (VLP) of the major capsid protein (L1) induce efficient production of antibodies, which confer protection against the same viral type. The methylotrophic yeast Pichia pastoris is an efficient and inexpensive expression system for the production of high levels of heterologous proteins stably using a wild-type gene in combination with an integrative vector. It was recently demonstrated that P. pastoris can produce the HPV-16 L1 protein by using an episomal vector associated with the optimized L1 gene. However, the use of an episomal vector is not appropriate for protein production on an industrial scale. In the present study, the vectors were integrated into the Pichia genome and the results were positive for L1 gene transcription and protein production, both intracellularly and in the extracellular environment. Despite the great potential for expression by the P. pastoris system, our results suggest a low yield of L1 recombinant protein, which, however, does not make this system unworkable. The achievement of stable clones containing the expression cassettes integrated in the genome may permit optimizations that could enable the establishment of a platform for the production of VLP-based vaccines.

Posible papel del factor de transcripción AP1 en la regulación tejido-específica del papilomavirus humano / Possible role of transcription factor AP1 in the tissue-specific regulation of human papillomavirus

Activator protein-1 (AP1) is a dimeric protein, consisting either of homodimers between c-Jun, JunB, and JunD of by heterodimers with members of the Fos-family by physically interacting via a "leucine zipper" region. AP1 is an important transcription factor initially identified as a DNA binding protein that bound to enhancer sequences of the human metallothionein IIA gene. The protein components of AP1 are encoded by a set of genes known as "immediate-early" genes that can be activated by a variety of growth factors and mitogens through several different signaling pathways. Until recently, AP1 was considered a transcription factor expressed in most tissues to regulate cellular and viral genes now, it is becoming evident that AP1 can be involved in tissue-specific regulation of target genes due to the differential combination of the components of this important transcription factor. AP1 plays a crucial role during human papillomavirus (HPV) early gene expression, in particular of the expression of E6 and E7 oncoproteins. The HPV are a group of DNA viruses consisting of more than 80 different genotypes. Some of these HPV, know as high risk HPV, are important etiologic agents of uterine-cervical cancer (CaCu). Of the different types of cancer, CaCu is one of the most frequent among women worldwide, constituting the second death cause due to neoplasia. During cellular transformation, HPV infect basal cells in stratified epithelium; their DNA integrate into the host genome usually through the E2 gene; as these cells differentiate and migrate into the upper layer of the epithelium, viral oncogene are expressed blocking their differentiation. Mutagenesis in AP1 sites belonging to the HPV promoter region (LCR) completely abolished the HPV promoter activity in different cell lines; these results and biochemistry assays on this AP1 transcription factor, that includes protein-protein interactions between AP1 and another factors as E7 from HPV, and YY-1; the post-translattional modification and, the retinoic acid interaction; suggest a role for this AP1 factor in tissue-specific transcription of the human papillomavirus.