A novel system for stable, high-level expression from the T7 promoter

Kęsik-Brodacka, Małgorzata; Romanik, Agnieszka; Mikiewicz, Diana; Płucienniczak, Grażyna; Płucienniczak, Andrzej

doi:10.1186/1475-2859-11-109

Cited by 21 publications

(16 citation statements)

References 13 publications

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“…The fragment was amplified using primers introducing NdeI and XhoI restriction sites at the 5’ and 3’ ends, respectively. The obtained fragment was inserted into an NdeI- and XhoI-digested pIGCmT7Kes expression plasmid [ 12 ] and transformed into the NM522 E . coli strain.…”

Section: Methodsmentioning

confidence: 99%

A novel hemagglutinin protein produced in bacteria protects chickens against H5N1 highly pathogenic avian influenza viruses by inducing H5 subtype-specific neutralizing antibodies

et al. 2017

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The highly pathogenic (HP) H5N1 avian influenza viruses (AIVs) cause a mortality rate of up to 100% in infected chickens and pose a permanent pandemic threat. Attempts to obtain effective vaccines against H5N1 HPAIVs have focused on hemagglutinin (HA), an immunodominant viral antigen capable of eliciting neutralizing antibodies. The vast majority of vaccine projects have been performed using eukaryotic expression systems. In contrast, we used a bacterial expression system to produce vaccine HA protein (bacterial HA) according to our own design. The HA protein with the sequence of the H5N1 HPAIV strain was efficiently expressed in Escherichia coli, recovered in the form of inclusion bodies and refolded by dilution between two chromatographic purification steps. Antigenicity studies showed that the resulting antigen, referred to as rH5-E. coli, preserves conformational epitopes targeted by antibodies specific for H5-subtype HAs, inhibiting hemagglutination and/or neutralizing influenza viruses in vitro. The proper conformation of this protein and its ability to form functional oligomers were confirmed by a hemagglutination test. Consistent with the biochemical characteristics, prime-boost immunizations with adjuvanted rH5-E. coli protected 100% and 70% of specific pathogen-free, layer-type chickens against challenge with homologous and heterologous H5N1 HPAIVs, respectively. The observed protection was related to the positivity in the FluAC H5 test (IDVet) but not to hemagglutination-inhibiting antibody titers. Due to full protection, the effective contact transmission of the homologous challenge virus did not occur. Survivors from both challenges did not or only transiently shed the viruses, as established by viral RNA detection in oropharyngeal and cloacal swabs. Our results demonstrate that vaccination with rH5-E. coli could confer control of H5N1 HPAIV infection and transmission rates in chicken flocks, accompanied by reduced virus shedding. Moreover, the role of H5 subtype-specific neutralizing antibodies in anti-influenza immunity and a novel correlate of protection are indicated.

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Section: Methodsmentioning

confidence: 99%

A novel hemagglutinin protein produced in bacteria protects chickens against H5N1 highly pathogenic avian influenza viruses by inducing H5 subtype-specific neutralizing antibodies

et al. 2017

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“…Its attractiveness for industrial applications results from its well‐understood genetics, cell biology, and easy handling. Expression systems based on E. coli allow for rapid growth, high product yield, cost‐effectiveness, easy process scale‐up, and short turnaround time . The limitations of this expression host for the production of complex recombinant biopharmaceuticals include the absence of mammalian‐like posttranslational modifications, such as glycosylation, phosphorylation, and proteolytic processing .…”

Section: Systems For the Production Of Biopharmaceuticalsmentioning

confidence: 99%

Progress in biopharmaceutical development

Kęsik-Brodacka

2017

Biotech and App Biochem

267

126

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Since its introduction in 1982, biopharmaceutical drugs have revolutionized the treatment of a broad spectrum of diseases and are increasingly used in nearly all branches of medicine. In recent years, the biopharmaceuticals market has developed much faster than the market for all drugs and is believed to have great potential for further dynamic growth because of the tremendous demand for these drugs. Biobetters, which contain altered active pharmaceutical ingredients with enhanced efficacy, will play an important role in the development of biopharmaceuticals. Another significant group of biopharmaceuticals are biosimilars. Their introduction in the European Union and, recently, the Unites States markets will reduce the costs of biopharmaceutical treatment. This review highlights recent progress in the field of biopharmaceutical development and issues concerning the registration of innovative biopharmaceuticals and biosimilars. The leading class of biopharmaceuticals, the current biopharmaceuticals market, and forecasts are also discussed.

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“…The T7 promoter is known to be the strongest promoter known for the prokaryotic expression of proteins 35 and in experimental E. coli cultures, consistently higher expression levels of the protein have been observed with the kanamycin marker than in cultures grown with the commonly used antibiotic ampicillin 36 with the target protein being almost 50% of total cellular protein in some cases. 37 It is possible to speculate that for the above reasons, we achieved high levels of RecA expression described in this study.…”

Section: Discussionmentioning

confidence: 99%

Cloning and high-level expression of Thermus thermophilus RecA in E. coli: purification and novel use in HBV diagnostics

Sundarrajan¹,

Rao²,

Padmanabhan³

2018

Brazilian Journal of Microbiology

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We studied the role of Thermus thermophilus Recombinase A (RecA) in enhancing the PCR signals of DNA viruses such as Hepatitis B virus (HBV). The RecA gene of a thermophilic eubacterial strain, T. thermophilus, was cloned and hyperexpressed in Escherichia coli. The recombinant RecA protein was purified using a single heat treatment step without the use of any chromatography steps, and the purified protein (>95%) was found to be active. The purified RecA could enhance the polymerase chain reaction (PCR) signals of HBV and improve the detection limit of the HBV diagnosis by real time PCR. The yield of recombinant RecA was ∼35 mg/L, the highest yield reported for a recombinant RecA to date. RecA can be successfully employed to enhance detection sensitivity for the diagnosis of DNA viruses such as HBV, and this methodology could be particularly useful for clinical samples with HBV viral loads of less than 10 IU/mL, which is interesting and novel.

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A novel system for stable, high-level expression from the T7 promoter

Cited by 21 publications

References 13 publications

A novel hemagglutinin protein produced in bacteria protects chickens against H5N1 highly pathogenic avian influenza viruses by inducing H5 subtype-specific neutralizing antibodies

A novel hemagglutinin protein produced in bacteria protects chickens against H5N1 highly pathogenic avian influenza viruses by inducing H5 subtype-specific neutralizing antibodies

Progress in biopharmaceutical development

Cloning and high-level expression of Thermus thermophilus RecA in E. coli: purification and novel use in HBV diagnostics

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