Search results for: T7 Epitope -Tag Antibody
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#23933075 2013/08/06 To Up
Display of the VP1 epitope of foot-and-mouth disease virus on bacteriophage T7 and its application in diagnosis.Foot-and-mouth disease (FMD) is a highly contagious epidemic disease threatening the cattle industry since the sixteenth century. In recent years, the development of diagnostic assays for FMD has benefited considerably from the advances of recombinant DNA technology. In this study, the immunodominant region of the capsid protein VP1 of the foot-and-mouth disease virus (FMDV) was fused to the T7 bacteriophage and expressed on the surface of the bacteriophage capsid protein. The recombinant protein of about 42 kDa was detected by the anti-T7 tag monoclonal antibody in Western blot analysis. Phage ELISA showed that both the vaccinated and positive infected bovine sera reacted significantly with the recombinant T7 particle. This study demonstrated the potential of the T7 phage displaying the VP1 epitope as a diagnostic reagent.
Chuan Loo Wong, Chin Chin Sieo, Wen Siang Tan
2534 related Products with: Display of the VP1 epitope of foot-and-mouth disease virus on bacteriophage T7 and its application in diagnosis.1 mg200 1mg100 100ug1-99 mg/ml/ea price x 210010 1 mg25100
#20600943 2010/06/20 To Up
Enhanced periplasmic expression of high affinity humanized scFv against Hepatitis B surface antigen by codon optimization.Production of properly folded, functional recombinant antibodies in a prokaryotic system is governed by multiple factors like codon usage, plasmid copy number, upstream elements such as leader sequence, mRNA stability and presence of tightly controlled promoters. Here we present a strategy for enhanced production of the functional scFv in Escherichia coli by codon optimization. We have previously reported the generation of humanized scFv form of a potentially neutralizing mouse monoclonal antibody (5S) to the Hepatitis B surface antigen. However, the expression level of 5S-scFv in E. coli was fairly low which was possibly due to the presence of rare codons. In the native 5S-scFv gene, almost 58% of codons showed poor codon bias with varying degrees of rare occurrence in the E. coli genes. We therefore designed a synthetic gene encoding the 5S-scFv protein by using E. coli preferred codon usage. The codon-optimized scFv gene was further cloned into a T7 expression system with a C-terminus His-tag and expressed as a soluble protein mainly in the periplasm. The scFv was both purified by IMAC and detected on Western blot with this His-tag. Using the codon optimization strategy, we were able to achieve a more than 100-fold increased periplasmic expression of soluble scFv. Further, the purified scFv was stable and retained its antigen-binding affinity and epitope specificity. Interestingly, based on secondary structure prediction, we observed that the mRNA secondary structure, including that of the 5'-end, may not have a significant role in the increased expression of this optimized gene.
Ashutosh Tiwari, Anurag Sankhyan, Navin Khanna, Subrata Sinha
2858 related Products with: Enhanced periplasmic expression of high affinity humanized scFv against Hepatitis B surface antigen by codon optimization.100 μg1 mg100ug100ug100μg96T2 Pieces/Box0.1ml
#20047046 2010/01/03 To Up
Exogenously expressed human Ku70 stabilizes Ku80 in Xenopus oocytes and induces heterologous DNA-PK catalytic activity.The Ku protein is a heterodimer composed of 70 kD (Ku70) and 80 kD (Ku80) subunits. Ku is the regulatory component of the DNA-dependent protein kinase (DNA-PK) that has a catalytic subunit of approximately 460 kD (DNA-PK(cs)). In this study, the two polypeptides (Ku80/Ku70) of the human Ku were expressed in Xenopus oocytes in order to investigate their over-expression, sub-cellular localization, and functional interaction with the Xenopus DNA-PK(cs). In vitro-transcribed mRNAs for Ku70 and Ku80 were obtained from the respective plasmid constructs. The exogenously expressed proteins from the injected mRNAs were immunoprecipitated using a specific anti-T7 Tag antibody. The T7 Tag epitope is present in the vector at the amino-terminus and is in-frame with the Ku cDNA sequences. While injected Ku70 mRNA translated to a full-length Ku70 polypeptide that translocated to the nucleus, injected Ku80 mRNA resulted in the expression of a truncated product that was retained in the cytoplasm. Although Ku80 mRNA was stable for a period of 18 h in the oocytes post-microinjection, the protein was only stabilized when co-expressed with Ku70, suggesting that Ku80 is susceptible to proteolytic degradation when not dimerized with Ku70. Furthermore, the immunocomplex was capable of phosphorylating the DNA-PK-specific substrate thereby indicating that the holoenzyme could functionally reconstitute in vivo in the oocytes by heterologous subunits thus demonstrating evolutionary conservation of the enzyme subunit structure and function among diverse species.
2104 related Products with: Exogenously expressed human Ku70 stabilizes Ku80 in Xenopus oocytes and induces heterologous DNA-PK catalytic activity.2 250 units100 μg2 mg96T10 2 50 ug100 mg10 ug25 10
#19243080 // To Up
Small epitope-linker modules for PCR-based C-terminal tagging in Saccharomyces cerevisiae.PCR-mediated gene modification is a powerful approach to the functional analysis of genes in Saccharomyces cerevisiae. One application of this method is epitope-tagging of a gene to analyse the corresponding protein by immunological methods. However, the number of epitope tags available in a convenient format is still low, and interference with protein function by the epitope, particularly if it is large, is not uncommon. To address these limitations and broaden the utility of the method, we constructed a set of convenient template plasmids designed for PCR-based C-terminal tagging with 10 different, relatively short peptide sequences that are recognized by commercially available monoclonal antibodies. The encoded tags are FLAG, 3 x FLAG, T7, His-tag, Strep-tag II, S-tag, Myc, HSV, VSV-G and V5. The same pair of primers can be used to construct tagged alleles of a gene of interest with any of the 10 tags. In addition, a six-glycine linker sequence is inserted upstream of these tags to minimize the influence of the tag on the target protein and maximize its accessibility for antibody binding. Three marker genes, HIS3MX6, kanMX6 and hphMX4, are available for each epitope. We demonstrate the utility of the new tags for both immunoblotting and one-step affinity purification of the regulatory particle of the 26S proteasome. The set of plasmids has been deposited in the non-profit plasmid repository Addgene (http://www.addgene.org).
Minoru Funakoshi, Mark Hochstrasser
1047 related Products with: Small epitope-linker modules for PCR-based C-terminal tagging in Saccharomyces cerevisiae.5 G96tests100.00 ug250 mg500 MG 1 G
#18406167 2008/03/05 To Up
Expression, purification and immunodetection of a recombinant fragment (residues 179-281) of the G protein from rabies virus ERA strain.The glycoprotein (G) of rabies virus (RV) is important for virus infectivity and induction of the protective immunity. In this study, the region comprising linear epitopes (residues 179-281, ERA strain), named rGERA179-281, was cloned in frame with a hexahistidine tag coding sequence at its N-terminal end and overexpressed in Escherichia coli Rosetta strain. The expression under transcriptional regulation of T7 promoter yielded insoluble protein aggregates in the form of inclusion bodies. The inclusion bodies were solubilized with 6M guanidine HCl and the protein was purified to homogeneity under denaturing conditions. Mass spectrometry data confirmed the identity of the protein. The purified protein (13.8kDa) showed significant reactivity with antibodies present in a therapeutic human rabies immune globulin (HRIG), as demonstrated by immunoblotting analysis. In addition, by in vitro competitive neutralization assay, rGERA179-281 led to a measurable reduction in the ability of HRIG to neutralize rabies virus. These results, along with the good yield obtained, encourage further studies on the more detailed immunological properties of rGERA179-281, such as the ability to induce rabies virus neutralizing antibodies and the production of anti-G monoclonal antibodies, which together, might be useful for the development of new diagnostic methods.
Enio J Bassi, Javier Vernal, Camila Zanluca, Hernán Terenzi, Carlos R Zanetti
2403 related Products with: Expression, purification and immunodetection of a recombinant fragment (residues 179-281) of the G protein from rabies virus ERA strain.1 mg25ml 1 mg1 mg10ml10001005ml1 mg1 mL100 100ul
#16481528 // To Up
Production of recombinant protein Pap31 and its application for the diagnosis of Bartonella bacilliformis infection.Tropical bartonellosis is a highly fatal epidemic and endemic infectious disease that occurs throughout the communities of the Andes Mountains in South America. The disease is caused by the facultative intracellular bacteria, Bartonella bacilliformis. The emergence of bartonellosis in new geographic areas and an increase in the number of reported cases suggest the need for a rapid test for epidemiologic study and investigation of the disease burden. The objective of this research is to develop a rapid serologic diagnostic test using recombinant antigens to overcome the limitations of the current standard IFA technique for laboratory diagnosis. Western blot analysis with patient sera of whole cell lysate separated on a 2D gel identified Pap31 as a dominant antigen. PCR primers were designed according to the sequence of ATCC strain 35685 to amplify the gene coding for Pap31 from a local isolate (HOSP 800-09, Peru). The amplicon was subsequently cloned into pET24a, adding the T7 tag, and expressed in E. coli. Patient sera with different IFA titers confirmed the diagnostic band of 31 kDa on a Western blot of SDS-PAGE. The performance of affinity-purified recombinant Pap31 (rPap31) was also evaluated in an ELISA format with 137 patient sera of known IFA titers. The range of ELISA reading from positive sera did not overlap with the range of those from negative sera, suggesting the potential application of rPap31 in both ELISA for high throughput regional hospital settings and in the construction of handheld rapid tests for rural clinical sites.
A Taye, H Chen, K Duncan, Z Zhang, L Hendrix, J Gonzalez, W Ching
1526 related Products with: Production of recombinant protein Pap31 and its application for the diagnosis of Bartonella bacilliformis infection.1mg100.1 mg2100ul0.1 mg10ìg52102
#11843180 // To Up
Ligand-mediated protection against phage lysis as a positive selection strategy for the enrichment of epitopes displayed on the surface of E. coli cells.We present a novel strategy, termed CISTEM, which allows direct in vivo screening of polypeptides displayed on the surface of E. coli cells by a combination of ligand-mediated protection and phage-mediated selection. The effectiveness of this new approach was demonstrated by displaying the T7.tag on the surface of E. coli as a fusion with the outer membrane protein A, the receptor for bacteriophage K3. A monoclonal T7.tag antibody was used as protective ligand for T7.tag-displaying cells and phage K3 for the elimination of unprotected cells. When populations of bacteria, containing between 6 to 10,000 cells displaying the T7.tag and approximately 10(8) cells displaying an unrelated OmpA fusion protein, were infected with phage K3, specific and antibody-dependent survival of T7.tag displaying cells was observed, yielding an enrichment factor of up to 10(7)-fold. The CISTEM technology was used to select sequences from a T7.tag-based, randomised library and the results were compared to those obtained from selection by MACS with the same library. Together, these results reveal a novel in vivo screening strategy in which an E. coli phage receptor is used as display plafform and selection is performed in suspension upon addition of a protective ligand and a bacteriophage. Extentions and modifications of the basic strategy should lead to novel applications for the identification of protein-ligand interactions.
P Camaj, A E Hirsh, W Schmidt, A Meinke, A von Gabain
2235 related Products with: Ligand-mediated protection against phage lysis as a positive selection strategy for the enrichment of epitopes displayed on the surface of E. coli cells.100 assays100.00 ul1 ml 100ul100 tests1,000 tests100.00 ul
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