Expression of the major capsid protein of human papillomavirus type 16 in Escherichia coli

Kelsall, Stephen R.; Kulski, Jerzy K.

doi:10.1016/0166-0934(95)00004-e

Cited by 11 publications

(8 citation statements)

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“…Though transient viral episome replication can occur in a number of in vitro cells (37), only keratinocytes, or cells with the potential for squamous maturation, can be productively infected, since viral capsid proteins are synthesized and virions are assembled only in terminally differentiated keratinocytes. PV capsid proteins, expressed in mammalian cells (61), insect cells (22), and E. coli (20,27), can be used to study virion assembly and DNA encapsidation (43,44,52,(57)(58)(59)(60). However, there remain large gaps in the understanding of PV life cycle.…”

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confidence: 99%

Saccharomyces cerevisiaeIs Permissive for Replication of Bovine Papillomavirus Type 1

Zhao

Frazer

2002

J Virol

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We recently demonstrated that Saccharomyces cerevisiae protoplasts can take up bovine papillomavirus type 1 (BPV1) virions and that viral episomal DNA is replicated after uptake. Here we demonstrate that BPV virus-like particles are assembled in infected S. cerevisiae cultures from newly synthesized capsid proteins and also package newly synthesized DNA, including full-length and truncated viral DNA and S. cerevisiae-derived DNA. Virus particles prepared in S. cerevisiae are able to convey packaged DNA to Cos1 cells and to transform C127 cells. Infectivity was blocked by antisera to BPV1 L1 but not antisera to BPV1 E4. We conclude that S. cerevisiae is permissive for the replication of BPV1 virus.Papillomaviruses (PVs) are exclusively epitheliotropic viruses and have evolved a unique replication strategy that depends upon the differentiation program of keratinocytes (15). Though transient viral episome replication can occur in a number of in vitro cells (37), only keratinocytes, or cells with the potential for squamous maturation, can be productively infected, since viral capsid proteins are synthesized and virions are assembled only in terminally differentiated keratinocytes. PV capsid proteins, expressed in mammalian cells (61), insect cells (22), and E. coli (20,27), can be used to study virion assembly and DNA encapsidation (43,44,52,(57)(58)(59)(60). However, there remain large gaps in the understanding of PV life cycle. Kreider et al. (24) first reported the use of athymic mouse xenograft culture to produce infectious human PV type 11 (HPV11) in vivo. In vitro raft culture systems have allowed differentiation-specific viral amplification, late gene expression, and virion morphogenesis for HPV31 (9, 46) and other PV types (2, 34). Recently, infectious particles have been produced (2,8,31,35,40), although the viral yield is generally small compared to input virions. However, only a small number of HPV types can be successfully grown in athymic and scid mouse xenograft systems or raft culture systems (55), and propagation of large numbers of viral particles in vitro is yet to be achieved (2).Lambert et al. (26) first used the S. cerevisiae system to study the expression and function of the bovine PV type 1 (BPV1) E2 gene. Dostatni et al. (5) used S. cerevisiae to express fulllength BPV1 E2 protein and assayed in vitro its capacity to modulate transcription. Prakash et al. (41) reported that BPV1 E2 protein regulates viral transcription by binding as a dimer to the DNA sequence ACCGN4CGGT. According to previous studies of viral DNA replication in yeast (21, 42), the basic requirements for viral cis and trans elements for episome replication are similar between S. cerevisiae and mammalian cells.We have recently observed that S. cerevisiae protoplasts, which have extensive endocytotic activity (10), can take up BPV1 virions, and the BPV1 episome can replicate (56). In the present study, we have studied whether S. cerevisiae exposed to PV virions can support production of infectious virions. MATERIALS AND METHO...

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confidence: 99%

Saccharomyces cerevisiaeIs Permissive for Replication of Bovine Papillomavirus Type 1

Zhao

Frazer

2002

J Virol

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“…These species are possibly degradation products of L1, since they were not seen in total extracts of P. pastoris that did not show expression of the recombinant protein. Other groups have also found degradation patterns after expression of the HPV16 L1 protein in insect cells [8], yeast [36] and bacteria [14, 15, 26, 37]. L1 was not detected in negative control extracts ( P. pastoris transformed with empty pPICHOLI vector, Fig.…”

Section: Resultsmentioning

confidence: 91%

“…Several approaches for expressing recombinant L1 from HPV16 have been tested using bacteria, e.g., Salmonella typhimurium [13], Escherichia coli [14, 15], Shigella flexneri [16], Lactobacillus casei [17], Lactococcus lactis [18], yeast, e.g., Saccharomyces cerevisiae [19–21], Schizosaccharomyces pombe [22], baculovirus-infected insect cells [23], transgenic plants, e.g., tobacco and potato [24], and mammalian cells [25]. Bacterial expression systems have proven to be quite limited in producing economically significant quantities of recombinant HPV-16 L1 VLPs [26].…”

Section: Introductionmentioning

confidence: 99%

Expression and characterization of HPV-16 L1 capsid protein in Pichia pastoris

et al. 2009

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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.

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“…Our western blot analysis revealed the presence of lower molecular weight protein species in the H. polymorpha crude cell lysates, but not in the P. pastoris KM71 crude cell lysates (Figure ). Several other groups have seen this phenomenon after expressing HPV16 L1 in insect cells (Kirnbauer et al, ), yeast (Bazan et al, ; Deschuyteneer et al, ; Jiang et al, ) and bacteria (Aires et al, ; Chen, Casini, Harrison, & Garcea, ; Kelsall & Kulski, ; Lai & Middelberg, ; Zhang et al, ). The lower molecular weight proteins are most probably degradation products of the full‐length L1 since they were not seen in total extracts of H. polymorpha before the induction of recombinant proteins (data not shown) (Bazan et al, ; Zhang et al, ).…”

Section: Discussionmentioning

confidence: 94%

Expression of unique chimeric human papilloma virus type 16 (HPV‐16) L1‐L2 proteins in Pichia pastoris and Hansenula polymorpha

Bredell

Smith

Görgens

et al. 2018

Yeast

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Cervical cancer is ranked the fourth most common cancer in women worldwide. Despite two prophylactic vaccines being commercially available, they are unaffordable for most women in developing countries. We compared the optimized expression of monomers of the unique HPV type 16 L1-L2 chimeric protein (SAF) in two yeast strains of Pichia pastoris, KM71 (Mut ) and GS115 (Mut ), with Hansenula polymorpha NCYC 495 to determine the preferred host in bioreactors. SAF was uniquely created by replacing the h4 helix of the HPV-16 capsid L1 protein with an L2 peptide. Two different feeding strategies in fed-batch cultures of P. pastoris Mut were evaluated: a predetermined feed rate vs. feeding based on the oxygen consumption by maintaining constant dissolved oxygen levels (DO stat). All cultures showed a significant increase in biomass when methanol was fed using the DO stat method. In P. pastoris the SAF concentrations were higher in the Mut strains than in the Mut strains. However, H. polymorpha produced the highest level of SAF at 132.10 mg L culture while P. pastoris Mut only produced 23.61 mg L . H. polymorpha showed greater potential for the expression of HPV-16 L1/L2 chimeric proteins despite the track record of P. pastoris as a high-level producer of heterologous proteins.

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Expression of the major capsid protein of human papillomavirus type 16 in Escherichia coli

Cited by 11 publications

References 41 publications

Saccharomyces cerevisiaeIs Permissive for Replication of Bovine Papillomavirus Type 1

Saccharomyces cerevisiaeIs Permissive for Replication of Bovine Papillomavirus Type 1

Expression and characterization of HPV-16 L1 capsid protein in Pichia pastoris

Expression of unique chimeric human papilloma virus type 16 (HPV‐16) L1‐L2 proteins in Pichia pastoris and Hansenula polymorpha

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