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

Coimbra, Eliane Campos; Gomes, Francisco Brás; Campos, Júlia Furtado; D’arc, Mirela; Carvalho, Jacqueline Campos Borba de; Mariz, Filipe Colaço; Jesus, André Luiz Santos de; Stocco, Rita de Cássia; Beçak, Willy; Freitas, Antonio Carlos de

doi:10.1590/s0100-879x2011007500141

Cited by 23 publications

(14 citation statements)

References 29 publications

(26 reference statements)

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“…In the colony blot assay, the plate with the recombinant colonies is incubated with methanol, for induction for 72 h. The PVDF membrane was laid on colonies of the transformants and incubated at 37 • C for 3 h. Following this, the membrane was washed with TTBS, blocked with 5% milk, and incubated with penta-His antibody (Qiagen, SP, Brazil) (1:1000) for 2 h at 28 • C. After further washing stage, the membrane was incubated with the anti-mouse HRP-conjugated antibody (1:2000) (Sigma-Aldrich, SP, Brazil) for 1 h. The reaction was revealed with tetramethylbenzidine (TMB) substrate solution (Sigma-Aldrich). In the case of the dot blot assays, the clones were cultivated on a small scale (5 mL) with YPD medium (1% yeast extract, 2% peptone, 2% glucose) for 24 h. At the end of this period, the clones were induced for heterologous expression by the addition of methanol to a final concentration of 0.5% over a period of 72 h, at 28 • C. The supernatant was recovered and used in the dot blot assay as described by Coimbra et al (2011) using the monoclonal anti-6xHis antibody conjugated with alkaline phosphatase (1:1000) (Sigma-Aldrich). The NTB/BCIP substrate was used for the revelation of reactions (Sigma-Aldrich).…”

Section: Screening Of the Recombinants (Colony Blot And Dot Blot)mentioning

confidence: 99%

Secretory expression of Porcine Circovirus Type 2 capsid protein in Pichia pastoris

Silva

Coimbra

Jesus

et al. 2014

Journal of Virological Methods

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Section: Screening Of the Recombinants (Colony Blot And Dot Blot)mentioning

confidence: 99%

Secretory expression of Porcine Circovirus Type 2 capsid protein in Pichia pastoris

Silva

Coimbra

Jesus

et al. 2014

Journal of Virological Methods

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“…The most common high-risk HPV types are 16 and 18 (Coimbra et al , 2011). To date, two prophylactic HPV vaccines are on the market, Gardasil ™ (Merck) and Cervarix ™ (Glaxo-SmithKline).…”

Section: Health Care Industrymentioning

confidence: 99%

“…Therefore, there is a great need for other strategies for the production of cheaper HPV vaccines that could be provided by public health programs, allowing a greater penetration into these communities (Bazan et al , 2009). 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 (Coimbra et al , 2011). It was recently demonstrated that P. pastoris could produce the HPV-16 L1 protein by using an episomal vector associated with the optimized L1 gene.…”

Section: Health Care Industrymentioning

confidence: 99%

“…In addition, the use of an integrative vector may favor the integration of more than one copy of the heterologous gene, which in many cases increases protein production (Clare et al , 1991). In Coimbra et al , 2011 studies, they integrated the vectors into the P. pastoris genome and the results were positive for L1 gene transcription and protein production, both intracellularly and in the extracellular environment. The problem was that their results suggest a low yield of L1 recombinant protein, but they admitted that they could still do better in the yield optimization process.…”

Section: Health Care Industrymentioning

confidence: 99%

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Applications of recombinant Pichia pastoris in the healthcare industry

Weinacker

Rabert

Zepeda

et al. 2013

Braz. J. Microbiol.

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Since the 1970s, the establishment and development of the biotech industry has improved exponentially, allowing the commercial production of biopharmaceutical proteins. Nowadays, new recombinant protein production is considered a multibillion-dollar market, in which about 25% of commercial pharmaceuticals are biopharmaceuticals. But to achieve a competitive production process is not an easy task. Any production process has to be highly productive, efficient and economic. Despite that the perfect host is still not discovered, several research groups have chosen Pichia pastoris as expression system for the production of their protein because of its many features. The attempt of this review is to embrace several research lines that have adopted Pichia pastoris as their expression system to produce a protein on an industrial scale in the health care industry.

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“…Since 2007, several investigators have reported the expression and purification of HPV L1 protein in Pichia pastoris, and characterized the system as a potential vaccine platform in view of its low cost and the high levels of recombinant protein expression reported in the last 15 years [35]. In a similar way, our research group has been working with the HPV -16, -18 and -33 L1 expression in P. pastoris [36,37].…”

Section: Attempts To Develop Alternatives For Prophylactic Hpv Vaccinesmentioning

confidence: 99%

Vaccine Strategies against Human Papillomavirus: A Discussion Focused on Developing Countries

Freitas¹

2012

J Clin Cell Immunol

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Cervical cancer is the second most common form of cancer among women, and responsible for 274,000 deaths each year, most of which occur in developing countries. Persistent infection with high-risk human papillomavirus (HR-HPV) is an essential factor in the development of cervical cancer and also a contributory factor in other types of cancer. The current prophylactic HPV vaccines provide protection against the -16 and -18 genotypes which are most commonly associated with cervical cancer worldwide. However, the increased costs of these vaccines inhibit their implementation in developing countries, affecting their viability. Moreover, a therapeutic vaccine is needed for women who are already infected by HPV and/or affected by HPV-related cancer. A number of innovative approaches to combat and treat HPV infection are currently being studied and some of these will be consider in this work, together with the development of new vaccines, especially in seriously affected areas located in developing countries. At the same time, there will be a discussion of the issues involved in carrying out effective HPV vaccination programs; these will take account of financial constraints, the lack of adequate infrastructure and the competing priorities, that are found in the surrounding social context of the developing countries.

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

Cited by 23 publications

References 29 publications

Secretory expression of Porcine Circovirus Type 2 capsid protein in Pichia pastoris

Secretory expression of Porcine Circovirus Type 2 capsid protein in Pichia pastoris

Applications of recombinant Pichia pastoris in the healthcare industry

Vaccine Strategies against Human Papillomavirus: A Discussion Focused on Developing Countries

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