Cloning, expression, purification and characterization of the cholera toxin B subunit and triple glutamic acid decarboxylase epitopes fusion protein in Escherichia coli

Gong, Zhaohui; Long, Xiang-E; Pan, Lin; Le, Yanping; Liu, Qiong; Wang, Shaomin; Guo, Junming; Xiao, Bingxiu; Zhou, Mi; Mei, Disen

doi:10.1016/j.pep.2009.04.002

Cited by 11 publications

(10 citation statements)

References 41 publications

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“…In addition, the protective effect against continued insulitis development was transferable to untreated NOD mice through transfer of CD4+ T cells from CTB-INS inoculated animals (Bergerot, et al, 1997). Our recent finding that CTB-INS is active in the suppression of DC maturation provides a possible explanation for CTB enhanced insulin mediated diabetes suppression observed in NOD mice studies employing CTB-INS fusion protein produced in viruses, plants, and silkworm larvae (Denes, et al, 2005, Gong, et al, 2007, Gong, et al, 2009). …”

Section: Discussionmentioning

confidence: 95%

Suppression of dendritic cell activation by diabetes autoantigens linked to the cholera toxin B subunit

et al. 2011

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Antigen presenting cells, specifically dendritic cells (DCs) are a focal point in the delicate balance between T cell tolerance and immune responses contributing to the onset of type I diabetes (T1D). Weak adjuvant proteins like the cholera toxin B subunit when linked to autoantigens may sufficiently alter the balance of this initial immune response to suppress the development of autoimmunity. To assess adjuvant enhancement of autoantigen mediated immune suppression of Type 1 diabetes, we examined the cholera toxin B subunit (CTB)-proinsulin fusion protein (CTB-INS) activation of immature dendritic cells (iDC) at the earliest detectable stage of the human immune response. In this study, Incubation of human umbilical cord blood monocyte-derived immature DCs with CTB-INS autoantigen fusion protein increased the surface membrane expression of DC toll-like receptor (TLR-2) while no significant upregulation in TLR-4 expression was detected. Inoculation of iDCs with CTB stimulated the biosynthesis of both CD86 and CD83 co-stimulatory factors demonstrating an immunostimulatory role for CTB in both DC activation and maturation. In contrast, incubation of iDCs with proinsulin partially suppressed CD86 co-stimulatory factor mediated DC activation, while incubation of iDCs with CTB-INS fusion protein completely suppressed iDC biosynthesis of both CD86 and CD83 costimulatory factors. The incubation of iDCs with increasing amounts of insulin did not increase the level of immune suppression but rather activated DC maturation by stimulating increased biosynthesis of both CD86 and CD83 costimulatory factors. Inoculation of iDCs with CTB-INS fusion protein dramatically increased secretion of the immunosuppressive cytokine IL-10 and suppressed synthesis of the pro-inflammatory cytokine IL12/23 p40 subunit protein suggesting that linkage of CTB to insulin (INS) may play an important role in mediating DC guidance of cognate naïve Th0 cell development into immunosuppressive T lymphocytes. Taken together, the experimental data suggests Toll like receptor 2 (TLR-2) plays a dominant role in CTB mediated INS inhibition of DC induced type 1 diabetes onset in human Type 1 diabetes autoimmunity. Further, fusion of CTB to the autoantigen was found to be essential for enhancement of immune suppression as codelivery of CTB and insulin did not significantly inhibit DC costimulatory factor biosynthesis. The experimental data presented supports the hypotheses that adjuvant enhancement of Author to whom correspondence should be addressed: William Langridge; Tel.: +1-909 558-1000 (81362); Fax: +1-909-558-0177 (Center for Health Disparities and Molecular Medicine, Loma Linda University, School of Medicine, Loma Linda, CA. 92354). Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its fina...

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Section: Discussionmentioning

confidence: 95%

Suppression of dendritic cell activation by diabetes autoantigens linked to the cholera toxin B subunit

et al. 2011

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“…Therefore, expression of the NIF and KGF mutant fusion protein is a practical strategy for preventing lung fibrosis. Recombinant NIF-KGF fusion protein has not previously been reported in either E. coli, insect, potato, tomato, or tobacco [24]. Here, we report the cloning and expression of the NIF-KGF mutant fusion gene in E. coli BL21 (DE3).…”

Section: Discussionmentioning

confidence: 98%

Protective effects of a bacterially expressed NIF–KGF fusion protein against bleomycin-induced acute lung injury in mice

Li¹,

Li²,

Zhang³

et al. 2010

ABBS

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Current evidence suggests that the keratinocyte growth factor (KGF) and the polymorphonuclear leukocyte may play key roles in the development of lung fibrosis. Here we describe the construction, expression, purification, and identification of a novel NIF (neutrophil inhibitory factor)-KGF mutant fusion protein (NKM). The fusion gene was ligated via a flexible octapeptide hinge and expressed as an insoluble protein in Escherichia coli BL21 (DE3). The fusion protein retained the activities of KGF and NIF, as it inhibited both fibroblast proliferation and leukocyte adhesion. Next, the effects of NKM on bleomycin-induced lung fibrosis in mice were examined. The mice were divided into the following four groups: (i) saline group; (ii) bleomycin group (instilled with 5 mg/kg bleomycin intratracheally); (iii) bleomycin plus dexamethasone (Dex) group (Dex was given intraperitoneally (i.p.) at 1 mg/kg/day 2 days prior to bleomycin instillation and daily after bleomycin instillation until the end of the treatment); and (iv) bleomycin plus NKM group (NKM was given i.p. at 2 mg/kg/day using the same protocol as the Dex group). NKM significantly improved the survival rates of mice exposed to bleomycin. The marked morphological changes and increased hydroxyproline levels resulted from the instillation of bleomycin (on Day 17) in the lungs were significantly inhibited by NKM. These results revealed that NKM can attenuate bleomycin-induced lung fibrosis, suggesting that NKM could be used to prevent bleomycin-induced lung damage or other interstitial pulmonary fibrosis.

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“…However, the expression of CTB protein in insects and plants is limited by low accumulation levels. Here, we report the cloning and expression of CTB protein in E. coli BL21 (Gong et al 2009). …”

Section: Discussionmentioning

confidence: 99%

“…When conjugated to autoantigens, CTB dramatically increases their tolerogenic potential after oral administration (Gong et al 2009;Sun et al 1994).…”

Section: Introductionmentioning

confidence: 99%

Cloning and expression of a cholera toxin beta subunit in Escherichia coli

2010

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Cholera is an acute infection of the intestine caused by theVibrio cholerae bacterium. This bacterium, a member of Vibrionaceae family, is a facultatively anaerobic, Gram-negative, non-spore-forming curved rod, about 1.4-2.6 µm long, and capable of both respiratory and fermentative metabolism. Cholera, characterized by numerous voluminous watery stools, is often accompanied by vomiting (leading to bicarbonate loss). One of the most important virulence factors of cholera is enterotoxin (ctxAB). The cholera toxin beta subunit (CTB) is a pentameric non-toxic portion of V. cholerae toxin, responsible for holotoxin binding to the GM1-ganglioside receptor that is present on most nucleated cells. When conjugated to autoantigens, CTB dramatically increases its tolerogenic potential after oral administration. In the present study, the CTB gene from a hypertoxigenic V. cholerae strain was amplified and cloned. We first amplified the CTB gene with specific primers and the Prime STAR enzyme. The amplified CTB gene was then cloned in pET-28 vector and introduced into Escherichia coli DH5α. pET plasmids containing the CTB gene plasmids were then extracted from the bacteria and used to transform an expression host (BL21). After culturing and induction of positive bacteria, SDS-PAGE and Western blotting for protein determination and verification were performed. We also quantified the level of recombinant CTB by ELISA. Our results show that, due to its rapid growth, an expression host such as E. coli can be useful for production of CTB protein. The ELISA results showed yields of recombinant protein of up to 1 mg/L culture. However, optimal conditions for expression in our study included choice of host strain, temperature used for culture, and concentration of antibiotic and inducer. Beta subunit has many important scientific applications. There is a great deal of interest in the use of CTB as an adjuvant for vaccines targeted for delivery to the mucosa-associated lymphatic tissues. The importance and multifunctional nature of Beta subunit of V. cholerae enterotoxin, e.g., in oral vaccine preparation, will make the use of prokaryotic systems (such as Escherichia coli) for production of this protein most advantageous.

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Cloning, expression, purification and characterization of the cholera toxin B subunit and triple glutamic acid decarboxylase epitopes fusion protein in Escherichia coli

Cited by 11 publications

References 41 publications

Suppression of dendritic cell activation by diabetes autoantigens linked to the cholera toxin B subunit

Suppression of dendritic cell activation by diabetes autoantigens linked to the cholera toxin B subunit

Protective effects of a bacterially expressed NIF–KGF fusion protein against bleomycin-induced acute lung injury in mice

Cloning and expression of a cholera toxin beta subunit in Escherichia coli

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Cloning, expression, purification and characterization of the cholera toxin B subunit and triple glutamic acid decarboxylase epitopes fusion protein in Escherichia coli

Cited by 11 publications

References 41 publications

Suppression of dendritic cell activation by diabetes autoantigens linked to the cholera toxin B subunit

Suppression of dendritic cell activation by diabetes autoantigens linked to the cholera toxin B subunit

Protective effects of a bacterially expressed NIF&ndash;KGF fusion protein against bleomycin-induced acute lung injury in mice

Cloning and expression of a cholera toxin beta subunit in Escherichia coli

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Protective effects of a bacterially expressed NIF–KGF fusion protein against bleomycin-induced acute lung injury in mice