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.

Expression of the bovine papillomavirus type 1, 2 and 4 L1 genes in the yeast Pichia pastoris

“…Enhancing expression levels in P. pastoris has required optimization of the heterologous gene through an increase in GC content and the replacement of rare codons with more frequent ones (Bazan et al, 2009;Kotzé et al, 2011). Eukaryotic genes are GC rich and thus more efficiently translated than are viral genes, which are generally rich in AT (Haas et al, 1996).…”

“…Significant levels of expression were detected only when the gene was expressed in a bioreactor. Conversely, Bazan et al (2009) have expressed a codon-optimized HPV16 L1 gene in P. pastoris using flasks; however, they applied a strategy using a non-integrative plasmid expression system. Unlike integrative systems, episomal plasmids have some important disadvantages such as clonal instability, which requires continuous antibiotic selection.…”

Relationship between human papilloma virus and benign and malign lesions of oral cavity and oropharynx, current approach

“…Birçok alt tip içeren uygun maliyetli aşı üretimi için çalışmalar sürmektedir. Burun spreyleri veya yama yolu gibi daha kolay uygulama yöntemlerinin geliştirilmesi, aynı zamanda daha kolay ve ucuz aşı erişilebilirliğine neden olabilir (56)(57)(58)(59) .…”

Non-Viral Delivery Systems in Gene Therapy and Vaccine Development

“…Other restriction is that the varieties of carbon sources that can be utilized by this species are limited [Bian et al, 2010]. Currently, two other species including Hansenula polymorpha and Pichia pastoris belonging to the Saccharomycetaceae family, could potentially overcome the described limitations of S. cerevisiae [Bazan et al, 2009;Bian et al, 2010]. On the other hand, these two species are broadly used as industrial platforms for heterologous protein production [Maleki et al, 2010;Bian et al, 2010].…”