High‐level expression and single‐step purification of recombinant <i>Bacillus anthracis</i> protective antigen from <i>Escherichia coli</i>

Lu, Jinbiao; Dong, Wei; Wang, Yefu; Wang, Guozhi

doi:10.1042/ba20070245

Cited by 8 publications

(5 citation statements)

References 26 publications

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“…E. coli provides an attractive alternative host to produce rPA and many researchers have expressed rPA from E. coli. However, such rPA always forms inclusion bodies in the cytoplasm of E. coli, [20][21][22][23][24] making difficult the procedure of its purification, and with which the biologically action of rPA might be different to natural PA. 9 To overcome this problem, a periplasmic expression strategy using secretion expression vectors had been well employed, 25,26 but relatively low amounts of soluble rPA are produced in the periplasm of E. coli compared with that in the cytoplasm of E. coli. In the present study, a new expression system including pTIG-Trx-PA vector and BL21 (DE3) was used, which supported a high expression of fully soluble rPA in the cytoplasm of E. coli without the need to resynthesize the PA gene with altered codon bias or fuse PA with GST 27 or PA4 with gIII protein of filamentous phage M13.…”

Section: Discussionmentioning

confidence: 99%

In vitro and in vivo activities of recombinant anthrax protective antigen co-expressed with thioredoxin inEscherichia coli

Zhu

et al. 2013

Human Vaccines & Immunotherapeutics

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Because of the central role it plays in the formation of lethal toxin and edema toxin, protective antigen (PA) is the principal target for the development of vaccines against anthrax. In the present study, we explored and compared the in vitro and in vivo activities of recombinant anthrax protective antigen (rPA) and receptor binding domain of protective antigen (PA4). As a result, the fully soluble rPA and PA4 proteins were successfully expressed in Escherichia coli by co-expression with thioredoxin (Trx), and the rPA was active in forming cytotoxic lethal toxins, indicating that the rPA protein retains a functionally biological activity. Furthermore, immunization with rPA protein induced stronger PA-specific immune responses in mice than PA4 protein. The protection elicited by immunization with PA4 suggests the presence of common neutralizing epitopes between rPA and PA4, but the immunization with rPA protein induced stronger neutralizing antibodies and protective levels against challenge with the B. anthracis strain A16R than the PA4 protein. The sera neutralizing antibodies titers correlated well with anti-PA group ELISA antibodies titers and the in vivo protective potency. Based on the results of cell cytotoxicity assays and the observed immune responses and protective potency, we concluded that the soluble rPA protein retains the in vitro and in vivo functionally biological activity and can be developed into a highly effective human subunit vaccine candidate against anthrax.

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Section: Discussionmentioning

confidence: 99%

In vitro and in vivo activities of recombinant anthrax protective antigen co-expressed with thioredoxin inEscherichia coli

Zhu

et al. 2013

Human Vaccines & Immunotherapeutics

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“…To overcome this problem. PA gene has been transformed into various hosts for the production of recombinant PA such as Esherichia coli [12][13][14] , Bacillus subtilis 15,16 , B. anthracis 17,18 , Bacillus brevis 11 , Baculovirus and Vaccinia Virus 19 , and Pseudomonas fluorescens 20 . However, each expression system has some limitations.…”

Section: Introductionmentioning

confidence: 99%

Production and Purification of Protective Antigen of Bacillus anthracis and Development of a Sandwich ELISA for its Detection

et al. 2020

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Anthrax, a zoonotic disease caused by Bacillus anthracis is important for biowarfare as well as public health point of view. The virulence factors of B. anthracis are encoded by the two plasmids, pXO1 and pXO2. Protective antigen (PA), an 83 kDa protein encoded by pXO1 along with lethal factor (LF, 90 kDa) or edema factor (EF, 89 kDa), makes the anthrax toxin responsible for causing the disease. Current detection and diagnostic systems for anthrax are mostly based on PA, a potential biomarker of B. anthracis. The objective of the present study was to produce and purify the PA for development of a sandwich ELISA for its detection. In this study, pYS5 plasmid containing the full PA gene was transformed into an 8 proteases deficient Bacillus anthracis host BH480. The PA was produced under shake flask conditions and purified using the gel filtration chromatography. The reactivity of PA with rabbit and mouse anti-PA antibodies was confirmed by Western blotting. The antibodies were purified and used for the development of a sandwich ELISA for detection of PA. The detection sensitivity of ELISA was found to be 3.9 ng/ mL PA.

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“…Numerous methods are described in the literature for the expression and isolation of PA, summarised in Table 2-1. PA has been generally expressed and isolated from B. anthracis (108,(112)(113)(114)(115)(116), B. subtilis (117,118), and E. coli (68,(119)(120)(121)(122)(123)(124)(125)(126)(127), but has also been successfully produced in a plant expression system (128). Whilst avirulent strains of B.…”

Section: Significancementioning

confidence: 99%

“…Although E. coli has limited capability to produce complex post-translational modifications or appropriate disulfide bonds (129), these limitations should not impact expression since PA does not appear to undergo any post-translational modifications nor does it contain disulfide bonds. E. coli expression of PA has been reported using plasmids that result in cytoplasmic (120)(121)(122)126) and periplasmic (68,117,119,125,127) localisation of the protein. Periplasmic targeting sequences are generally used to express proteins that require the oxidising environment of the periplasm for appropriate folding and disulfide bond formation (130) or to express proteins that can be toxic when accumulated in the cytoplasm (131), but may also reduce protein degradation and improve soluble protein extraction (129).…”

Section: Significancementioning

confidence: 99%

“…Yields up to 0.5 mg of PA per litre of culture were reported (124). More recent attempts to express PA localised to the cytoplasm have reported significantly higher yields than periplasmic localisation (121,122,126). Whilst isolation of PA from E. coli cytoplasmic proteins is more difficult, the addition of affinity tags such as 6xHis have been demonstrated to aid purification (120,121,126).…”

Section: Significancementioning

confidence: 99%

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B. anthracis protective antigen: a molecular imaging agent targeting squamous cell carcinoma

Crawford¹

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Targeted molecular imaging is an emerging non-invasive tool for the identification and characterisation of cancers based on physiological and molecular properties. Molecular imaging is likely to play a significant role in the development and validation of future biomarkers due to the ability to identify and monitor real time tumour characteristics noninvasively. There is currently a need to identify novel biomarkers that can be used to target, or inform on, tumour specific processes. The human anthrax toxin receptors Tumour Endothelial Marker 8 (TEM8) and Capillary Morphogenesis Gene 2 (CMG2) have been identified as potential novel tumour biomarkers. TEM8 and CMG2 are cell surface receptors involved in angiogenesis and extracellular matrix homeostasis, processes that are important for tumour growth and metastasis. TEM8 is particularly interesting due to its tumour specific upregulation and undetectable expression in many healthy adult tissues. One method to target these receptors is to exploit their exogenous ligand Protective Antigen (PA). PA is a protein produced by the bacterium B. anthracis that mediates binding and internalisation of toxins into cells. Whilst preclinical studies have shown that PA can be used to specifically target toxins to tumours, the tumour specific localisation of PA in vivo has yet to be demonstrated. We have expressed recombinant PA, using an E. coli expression system, and purified the protein by liquid chromatography techniques. PA was non-specifically labelled with an amine reactive near infra-red fluorescent dye for optical imaging or 89 Zr-Desferoxamine (89 Zr-DFO) for positron emission tomography (PET). The labelled PA was intravenously (IV) injected into female HPV38E6E7-FVB mice, a transgenic mouse model of squamous cell carcinoma (SCC) that endogenously produces tumours in response to repeated UV irradiation. The fluorescently labelled PA protein was highly specific to the SCC tumours, resulting in rapid tumour uptake and exclusive tumour identification 24 hours post injection. Post mortem analysis, 48 hours post injection, showed that the fluorescence in the tumour was significantly higher (p < 0.002, two tailed paired T test) than the adjacent skin, the liver, and the kidneys. Immunofluorescence analysis of formalin-fixed paraffin-embedded skin Publications included in this thesis No publications included.

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High‐level expression and single‐step purification of recombinant Bacillus anthracis protective antigen from Escherichia coli

Cited by 8 publications

References 26 publications

In vitro and in vivo activities of recombinant anthrax protective antigen co-expressed with thioredoxin inEscherichia coli

In vitro and in vivo activities of recombinant anthrax protective antigen co-expressed with thioredoxin inEscherichia coli

Production and Purification of Protective Antigen of Bacillus anthracis and Development of a Sandwich ELISA for its Detection

B. anthracis protective antigen: a molecular imaging agent targeting squamous cell carcinoma

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