scholarly journals pC6-2/caspase-6 system to purify glutathione-S-transferase-free recombinant fusion proteins expressed in Escherichia coli

2006 ◽  
Vol 1 (4) ◽  
pp. 1820-1827 ◽  
Author(s):  
Prabhat Kumar Purbey ◽  
P Cyril Jayakumar ◽  
Milind S Patole ◽  
Sanjeev Galande
Keyword(s):  
Escherichia Coli ◽  
Fusion Proteins ◽  
Glutathione S Transferase ◽  
Recombinant Fusion Proteins ◽  
Caspase 6

An ELISA sandwich capture assay for recombinant fusion proteins containing glutathione-S-transferase

1992 ◽  
Vol 156 (1) ◽  
pp. 101-105 ◽  
Author(s):  
Daniel U. Rabin ◽  
Rebecca Palmer-Crocker ◽  
Diane V. Mierz ◽  
Kwok K. Yeung
Keyword(s):  
Fusion Proteins ◽  
Glutathione S Transferase ◽  
Recombinant Fusion Proteins ◽  
Capture Assay

scholarly journals Monoclonal Antibodies to Escherichia coli-Expressed P46 and P65 Membranous Proteins for Specific Immunodetection of Mycoplasma hyopneumoniae in Lungs of Infected Pigs

2003 ◽  
Vol 10 (3) ◽  
pp. 459-468 ◽  
Author(s):  
K. Cheikh Saad Bouh ◽  
F. Shareck ◽  
S. Dea
Keyword(s):  
Escherichia Coli ◽  
Monoclonal Antibodies ◽  
Fusion Proteins ◽  
Genomic Sequence ◽  
Clinical Signs ◽  
Mycoplasma Hyopneumoniae ◽  
Mycoplasma Species ◽  
Antigenic Determinants ◽  
Recombinant Fusion Proteins ◽  
Species Specific

ABSTRACT The P46 and P65 proteins of Mycoplasma hyopneumoniae are two membranous proteins carrying species-specific antigenic determinants. Based on the genomic sequence of the reference strain ATCC 25934, primers were designed for PCR amplification of the genes encoding entire P46 (1,260 bp) and P65 (1,803 bp) and N-terminally truncated P65c (1,200 bp). These primers were shown to be specific to M. hyopneumoniae since no DNA amplicons could be obtained with other mycoplasma species that commonly colonize the porcine respiratory tract. Both amplified genes were then cloned into the pGEX-4T-1 vector to be expressed in Escherichia coli cells as recombinant fusion proteins with glutathione S-transferase (GST). Prior to generation of expression constructs, TGA nonsense codons, exceptionally used for tryptophan residues by M. hyopneumoniae, had been converted to TGG codons by PCR-directed mutagenesis. Following induction by IPTG (isopropyl-β-d-thiogalactopyranoside), both GST-P46 and GST-P65c recombinant fusion proteins were recovered by disrupting transformed cells by sonication, purified by affinity chromatography, and then cut with thrombin to release the P46 and P65c moieties. The enriched E. coli-expressed P46 and P65c proteins were used to immunize female BALB/c mice for the generation of anti-P46 and anti-P65c monoclonal antibodies (MAbs). The polypeptide specificities of MAbs obtained was confirmed by Western blotting with cell lysates prepared from the homologous strain. Cross-reactivity study of the anti-P46 and anti-P65c MAbs towards two other M. hyopneumoniae reference strains (ATCC 25095 and J strains) and Quebec field strains that had been isolated in culture, suggested that the MAbs obtained against both membranous proteins were directed against highly conserved species-specific epitopes. No reactivity to other mycoplasma species tested was demonstrated. Clinical signs and lesions suggestive of enzootic pneumonia were reproduced in specific-pathogen-free pigs that had been inoculated intratracheally with a virulent Quebec field strain (IAF-DM9827) of M. hyopneumoniae. Both anti-P46 and anti-P65c MAbs permitted effective detection by indirect immunofluorescence and indirect immunoperoxidase assay of M. hyopneumoniae in, respectively, frozen and formalin-fixed, paraffin-embedded lung sections from pigs that were killed after the 6- to 7-week observation period.

scholarly journals Expression in Bacteria of the Gene Encoding the gp43 Antigen of Paracoccidioides brasiliensis: Immunological Reactivity of the Recombinant Fusion Proteins

2002 ◽  
Vol 9 (6) ◽  
pp. 1200-1204 ◽  
Author(s):  
Susana N. Diniz ◽  
Kátia C. Carvalho ◽  
Patrícia S. Cisalpino ◽  
José F. Silveira ◽  
Luiz R. Travassos ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Fusion Protein ◽  
Fusion Proteins ◽  
Inclusion Bodies ◽  
Paracoccidioides Brasiliensis ◽  
Immunological Reactivity ◽  
Gene Encoding ◽  
Recombinant Fusion Proteins ◽  
Gst Fusion Protein

ABSTRACT gp43 is the major diagnostic antigen of Paracoccidioides brasiliensis, the agent of paracoccidioidomycosis (PCM) in humans. In the present study, cDNA of the gp43 gene (PbGP43) was obtained by reverse transcriptase PCR, inserted into a pGEX vector in frame with the glutathione S-transferase (GST) gene, and expressed in Escherichia coli as inclusion bodies. Immunoblotting showed that all sera from patients with chronic pulmonary and acute lymphatic forms of PCM reacted with the recombinant fusion protein of the mature gp43 (381 amino acids). Reactivity with fusion proteins containing subfragments of the N-terminal, internal, or C-terminal regions occurred eventually, and the C-terminal region was the most antigenic. Lack of reactivity with the subfragments may be due to the conformational nature of the gp43 epitopes. Sera from patients with aspergillosis, candidiasis, and histoplasmosis did not react with the gp43-GST fusion protein. Our results suggest that recombinant gp43 corresponding to the processed antigen can be a useful tool in the diagnosis of PCM.

Affinity purification of insoluble recombinant fusion proteins containing glutathione-S-transferase

1992 ◽  
Vol 39 (8) ◽  
pp. 828-832 ◽  
Author(s):  
John Hartman ◽  
Paru Daram ◽  
Raymond A. Frizzell ◽  
Thomas Rado ◽  
Dale J. Benos ◽  
...  
Keyword(s):  
Fusion Proteins ◽  
Affinity Purification ◽  
Glutathione S Transferase ◽  
Recombinant Fusion Proteins

scholarly journals Antibodies to the HMW1/HMW2 and Hia Adhesins of Nontypeable Haemophilus influenzae Mediate Broad-Based Opsonophagocytic Killing of Homologous and Heterologous Strains

2014 ◽  
Vol 21 (5) ◽  
pp. 613-621 ◽  
Author(s):  
Linda E. Winter ◽  
Stephen J. Barenkamp
Keyword(s):  
Escherichia Coli ◽  
Haemophilus Influenzae ◽  
Fusion Proteins ◽  
Phagocytic Cell ◽  
Nontypeable Haemophilus Influenzae ◽  
Content Type ◽  
Recombinant Fusion Proteins ◽  
Large Panel ◽  
Opsonophagocytic Killing ◽  
Surface Adhesins

ABSTRACTThe HMW1/HMW2 and Hia proteins are highly immunogenic surface adhesins of nontypeableHaemophilus influenzae(NTHi). Approximately 75% of NTHi strains express HMW1/HMW2 adhesins, and most of the remaining 25% express an Hia adhesin. Our objective in this study was to assess the ability of antisera raised against purified HMW1/HMW2 proteins or recombinant Hia proteins to mediate opsonophagocytic killing of a large panel of unrelated NTHi strains. Native HMW1/HMW2 proteins were purified from three HMW1/HMW2-expressing NTHi strains. Recombinant fusion proteins expressing surface-exposed segments of either of two prototype Hia proteins were purified fromEscherichia colitransformants. Immune sera raised in guinea pigs were assessed for their ability to mediate killing of NTHi in an opsonophagocytic assay with the HL-60 phagocytic cell line. The three HMW1/HMW2 antisera mediated killing of 22 of 65, 43 of 65, and 28 of 65 unrelated HMW1/HMW2-expressing NTHi strains, respectively. As a group, the three sera mediated killing of 48 of 65 HMW1/HMW2-expressing strains. The two Hia immune sera mediated killing of 12 of 24 and 13 of 24 unrelated Hia-expressing NTHi strains, respectively. Together, they mediated killing of 15 of 24 Hia-expressing strains. Neither the HMW1/HMW2 nor the Hia antisera mediated killing of NTHi expressing the alternative adhesin type. Antibodies directed against native HMW1/HMW2 proteins and recombinant Hia proteins are capable of mediating broad-based opsonophagocytic killing of homologous and heterologous NTHi strains. A vaccine formulated with a limited number of HMW1/HMW2 and Hia proteins might provide protection against disease caused by most NTHi strains.

Expression of a Fusion Protein of Human Proinsulin with Glutathione-S-Transferase in Escherichia coli

2014 ◽  
Vol 998-999 ◽  
pp. 248-251
Author(s):  
Zhi Xin Di ◽  
Jian Zhong Ma ◽  
Yong Gang Wang
Keyword(s):  
Escherichia Coli ◽  
Fusion Protein ◽  
Fusion Proteins ◽  
Inclusion Bodies ◽  
Glutathione S Transferase ◽  
E Coli ◽  
Human Proinsulin ◽  
Sds Page ◽  
Dna Fragment ◽  
Sequence Encoding

A DNA sequence encoding for the human proinsulin was designed according to the codon bias of Escherichia coli and then chemically synthesized. The synthesized DNA fragment was subcloned into pGEX-3X for expression in E. coli BL21 (DE3) and E. coli BL21 Star (DE3), respectively. Conditions for the highest expression of the GST-proinsulin fusion proteins were optimized. These conditions are that cells of E. coli BL21 star (DE3) are incubated in 100mL of the LB medium with 2 mmol/L IPTG and 60μ?g/mL ampicillin at 26oCfor 4h. After disrupted E. coli cells with ultrasonication, inclusion bodies were precipitated from cell lysis and washed. Fusion proteins from the inclusion bodies were redissolved in 8mmol/L of urea. After dialysed in purified water, fusion proteins were analysed by SDS-PAGE. The purity of the fusion protein is about 80.5% in total. The fusion protein from SDS-PAGE was further identified by mass/mass spectrum. GST in the dyad protein is confirmed by the 9 matched sequences. However, the left part is proved a polypeptide of which is completely different from the human proinsulin.

scholarly journals Polyclonal Antibodies to Glutathione S-Transferase- Verotoxin Subunit A Fusion Proteins Neutralize Verotoxins

2002 ◽  
Vol 9 (3) ◽  
pp. 687-692 ◽  
Author(s):  
P. H. M. Leung ◽  
J. S. M. Peiris ◽  
W. W. S. Ng ◽  
W. C. Yam
Keyword(s):  
Escherichia Coli ◽  
Sequence Homology ◽  
Fusion Proteins ◽  
Polyclonal Antibodies ◽  
Glutathione S Transferase ◽  
Subunit A ◽  
Immunological Diagnosis ◽  
Cross Neutralization ◽  
Transmembrane Regions ◽  
Gst Fusion Proteins

ABSTRACT The A1 subunits of verotoxin-1 (VT1) and VT2 genes were cloned into pGEX-4T-2 for the expression of glutathione S-transferase (GST) fusion proteins. The N-terminal and the transmembrane regions of the A1 subunits were excluded from the constructs in order to increase the product yields. Polyclonal anti-VT1A1 and anti-VT2A1 antibodies were produced by immunizing rabbits with GST-VT1A1 and GST-VT2A1 fusion proteins, respectively. The antibodies were tested for their ability to neutralize active toxins from 45 VT-producing Escherichia coli (VTEC) strains. The antibodies had significantly high neutralizing activities against their homologous toxins. The average percentages of neutralization of VT1 by anti-GST-VT1A1 and anti-GST-VT2A1 were 76.7% ± 7.9% and 3.6% ± 2.3%, respectively, and those of VT2 were 1.7% ± 2.3% and 82.5% ± 13.9%, respectively. VT2 variant toxin was neutralized by anti-GST-VT2A1, with cross neutralization being a possible consequence of sequence homology between VT2 and a VT2 variant. To our knowledge, this is the first report on the production of polyclonal antibodies from GST-VT fusion proteins. The antibodies were shown to exhibit specific toxin neutralizing activities and may be useful for immunological diagnosis of VTEC infections.

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