A Nonfusogenic Antigen Mimic of Influenza Hemagglutinin Glycoproteins Constituted with Soluble Full-Length HA1 and Truncated HA2 Proteins Expressed in E. coli

2014 ◽  
Vol 57 (2) ◽  
pp. 128-137
Author(s):  
Chang Sup Kim ◽  
Youn-Je Park
Keyword(s):  
Full Length ◽  
E Coli ◽  
Influenza Hemagglutinin

scholarly journals Expression Analysis and Nuclear Import Study of Full-length Isoforms Importin α as 6x Histidin-tagged Fusion Protein on the Intracellular Localization of Recombinant HBV Core Protein

2015 ◽  
Vol 10 (1) ◽  
Author(s):  
Aris Haryanto
Keyword(s):  
Fusion Protein ◽  
Nuclear Import ◽  
Recombinant Dna ◽  
Core Protein ◽  
Intracellular Localization ◽  
Full Length ◽  
Nuclear Pore ◽  
E Coli ◽  
Importin Α ◽  
Localization Signal

Isoform importin α molecules play a central role in the classical nuclear import pathway, that occurs throughthe nuclear pore complex (NPC) and typically requires a specific nuclear localization signal (NLS). In this study,it was investigated the role of isoforms importin α in the nuclear import of wild type recombinant hepatitis B viruscore protein (WT rHBc), phosphorylated recombinant HBV core (rHBc) and recombinant HBV core without NLSby co-immunoprecipitation. Four recombinant full-length isoforms importin α as 6x histidin-tagged fusion proteinwere expressed and analysed from expression plasmid vectors Rch1, pHM 1969, pHM 1967 and pHM 1965. Theresults indicated that importin α-1, importin α-3, importin α-4 and importin α-5 can be expressed and isolatedfrom E. coli transformed recombinant DNA plasmid as protein in size around 58-60 kDa. By the nuclear transportstudy shown that isoforms importin α are involved in the nuclear import of WT rHBc, phosphorylated rHBc andrHBc without NLS. It also indicated that they have an important role for nuclear transport of from cytoplasm intothe nucleus.Keywords: NPC, NLS, importin α, importin β, isoforms importin α as 6x histidin-tagged fusion protein, WTrHBc, SV40 Tag, co-immunoprecipitation, westernblotting.

scholarly journals Expression and characterization of recombinant fungal acetyl-CoA carboxylase and isolation of a soraphen-binding domain

2004 ◽  
Vol 380 (1) ◽  
pp. 105-110 ◽  
Author(s):  
Stephanie C. WEATHERLY ◽  
Sandra L. VOLRATH ◽  
Tedd D. ELICH
Keyword(s):  
Magnaporthe Grisea ◽  
Fatty Acid Biosynthesis ◽  
Recombinant Expression ◽  
Phytopathogenic Fungi ◽  
Full Length ◽  
Primary Metabolism ◽  
Acetyl Coa Carboxylase ◽  
Acetyl Coa ◽  
E Coli ◽  
High Affinity

Acetyl-CoA carboxylase (ACC) catalyses the first step in fatty-acid biosynthesis. Owing to its role in primary metabolism, ACC has been exploited as a commercial herbicide target and identified as a chemically validated fungicide target. In animals, ACC is also a key regulator of fat metabolism. This function has made ACC a prime target for the development of anti-obesity and anti-Type II diabetes therapeutics. Despite its economic importance, there is a lack of published information on recombinant expression of ACC. We report here the expression of enzymically active fungal (Ustilago maydis) ACC in Escherichia coli. The recombinant enzyme exhibited Km values of 0.14±0.013 mM and 0.19±0.041 mM for acetyl-CoA and ATP respectively, which are comparable with those reported for the endogenous enzyme. The polyketide natural product soraphen is a potent inhibitor of the BC (biotin carboxylase) domain of endogenous fungal ACC. Similarly, recombinant ACC activity was inhibited by soraphen with a Ki of 2.1±0.9 nM. A truncated BC domain that included amino acids 2–560 of the full-length protein was also expressed in E. coli. The isolated BC domain was expressed to higher levels, and was more stable than full-length ACC. Although incapable of enzymic turnover, the BC domain exhibited high-affinity soraphen binding (Kd 1.1±0.3 nM), demonstrating a native conformation. Additional BC domains from the phytopathogenic fungi Magnaporthe grisea and Phytophthora infestans were also cloned and expressed, and were shown to exhibit high-affinity soraphen binding. Together, these reagents will be useful for structural studies and assay development.

Cloning and high expression of full length hTRF1 in E. Coli

2001 ◽  
Vol 13 (3) ◽  
pp. 179-180
Author(s):  
He Huang ◽  
Parwaresh R ◽  
Kellner U ◽  
Qiao-fang Chen ◽  
Jie Sun
Keyword(s):  
Full Length ◽  
High Expression ◽  
E Coli

scholarly journals Towards Designer Cellulosomes in Clostridia: Mannanase Enrichment of the Cellulosomes Produced by Clostridium cellulolyticum

2004 ◽  
Vol 186 (19) ◽  
pp. 6544-6552 ◽  
Author(s):  
Stéphanie Perret ◽  
Anne Bélaich ◽  
Henri-Pierre Fierobe ◽  
Jean-Pierre Bélaich ◽  
Chantal Tardif
Keyword(s):  
Guar Gum ◽  
Specific Activity ◽  
Full Length ◽  
Expression Vectors ◽  
Leader Peptide ◽  
Crystalline Cellulose ◽  
Glycosyl Hydrolases ◽  
Truncated Form ◽  
E Coli ◽  
Clostridium Cellulolyticum

ABSTRACT The man5K gene of Clostridium cellulolyticum was cloned and overexpressed in Escherichia coli. This gene encodes a 424-amino-acid preprotein composed of an N-terminal leader peptide, followed by a dockerin module and a C-terminal catalytic module belonging to family 5 of the glycosyl hydrolases. Mature Man5K displays 62% identity with ManA from Clostridium cellulovorans. Two forms of the protein were purified from E. coli; one form corresponds to the full-length enzyme (45 kDa), and a truncated form (39 kDa) lacks the N-terminal dockerin module. Both forms exhibit the same typical family 5 mannanase substrate preference; they are very active with the galactomannan locust bean gum, and the more galacto-substituted guar gum molecules are degraded less. The truncated form, however, displays fourfold-higher activity with galactomannans than the full-length enzyme. Man5K was successfully overproduced in C. cellulolyticum by using expression vectors. The trans-produced protein was found to be incorporated into the cellulosomes and became one of the major enzymatic components. Modified cellulosomes displayed 20-fold-higher specific activities than control fractions on galactomannan substrates, whereas the specific activity on crystalline cellulose was reduced by 20%. This work clearly showed that the composition of the cellulosomes is obviously regulated by the relative amounts of the enzymes produced and that this composition can be engineered in clostridia by structural gene cloning.

scholarly journals M2 Proton Channel Structural Validation from Full-Length Protein Samples in Synthetic Bilayers and E. coli Membranes

2012 ◽  
Vol 124 (33) ◽  
pp. 8508-8511 ◽  
Author(s):  
Yimin Miao ◽  
Huajun Qin ◽  
Riqiang Fu ◽  
Mukesh Sharma ◽  
Thach V. Can ◽  
...  
Keyword(s):  
Full Length ◽  
Proton Channel ◽  
E Coli ◽  
M2 Proton Channel ◽  
Structural Validation

scholarly journals M2 Proton Channel Structural Validation from Full-Length Protein Samples in Synthetic Bilayers and E. coli Membranes

2012 ◽  
Vol 51 (33) ◽  
pp. 8383-8386 ◽  
Author(s):  
Yimin Miao ◽  
Huajun Qin ◽  
Riqiang Fu ◽  
Mukesh Sharma ◽  
Thach V. Can ◽  
...  
Keyword(s):  
Full Length ◽  
Proton Channel ◽  
E Coli ◽  
M2 Proton Channel ◽  
Structural Validation

scholarly journals Predicting the strength of UP-elements and full-length E. coli σE promoters

2011 ◽  
Vol 40 (7) ◽  
pp. 2907-2924 ◽  
Author(s):  
Virgil A. Rhodius ◽  
Vivek K. Mutalik ◽  
Carol A. Gross
Keyword(s):  
Full Length ◽  
E Coli ◽  
Up Elements

scholarly journals CEACAM1 Activation by CbpF-Expressing E. coli

2021 ◽  
Vol 11 ◽  
Author(s):  
Amjad Shhadeh ◽  
Johanna Galaski ◽  
Tamar Alon-Maimon ◽  
Jamal Fahoum ◽  
Reuven Wiener ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Tumor Immunity ◽  
Nk Cell ◽  
Fusobacterium Nucleatum ◽  
Full Length ◽  
Inhibitory Receptors ◽  
Negative Bacterium ◽  
Gram Negative ◽  
E Coli ◽  
Trimeric Autotransporter Adhesin

Recent studies on the oral, anaerobic, gram-negative bacterium Fusobacterium nucleatum revealed its presence and involvement in colorectal, esophageal and breast cancer. We previously demonstrated that F. nucleatum binds and activates the human inhibitory receptors TIGIT and CEACAM1 leading to inhibition of T and NK cell anti-tumor immunity. CEACAM1 was found to be bound and activated by the fusobacterial trimeric autotransporter adhesin CbpF. Here we report the generation of a recombinant E. coli expressing full-length CbpF that efficiently binds and activates CEACAM1.

Purification and functional characterization of the full length recombinant Ebola virus VP35 protein expressed in E. coli

2009 ◽  
Vol 66 (1) ◽  
pp. 113-119 ◽  
Author(s):  
Luca Zinzula ◽  
Francesca Esposito ◽  
Elke Mühlberger ◽  
Martina Trunschke ◽  
Dominik Conrad ◽  
...  
Keyword(s):  
Ebola Virus ◽  
Functional Characterization ◽  
Full Length ◽  
E Coli

Biophysical Characterization of Interaction between E. coli Alanyl-tRNA Synethase with its Promoter DNA

2020 ◽  
Vol 27 (7) ◽  
pp. 635-648
Author(s):  
Baisakhi Banerjee ◽  
Sayak Ganguli ◽  
Rajat Banerjee
Keyword(s):  
Dna Binding ◽  
Analytical Ultracentrifugation ◽  
Isothermal Calorimetry ◽  
Trna Synthetase ◽  
Full Length ◽  
Sedimentation Equilibrium ◽  
Titration Calorimetry ◽  
E Coli ◽  
Promoter Dna

Background: Aminoacyl-tRNA Synthetases (aaRSs) are well known for their role in the translation process. Lately investigators have discovered that this family of enzymes are also capable of executing a broad repertoire of functions that not only impact protein synthesis, but extend to a number of other activities. Till date, transcriptional regulation has so far only been described in E. coli Alanyl-tRNA synthetase and it was demonstrated that alaRS binds specifically to the palindromic DNA sequence flanking the gene’s transcriptional start site and thereby regulating its own transcription. Objective: In the present study, we have characterized some of the features of the alaRS-DNA binding using various biophysical techniques. Methods: To understand the role of full length protein and oligomerization of alaRS in promoter DNA binding, two mutants were constructed, namely, N700 (a monomer, containing the N-terminal aminoacylation domain but without the C-terminal part) and G674D (previously demonstrated to form full-length monomer). Protein-DNA binding study using fluorescence spectroscopy, analytical ultracentrifugation, Isothermal Titration Calorimetry was conducted. Results: Sedimentation equilibrium studies clearly demonstrated that monomeric variants were unable to bind promoter DNA. Isothermal Calorimetry (ITC) experiment was employed for further characterization of wild type alaRS-DNA interaction. It was observed that full length E. coli Alanyl-tRNA synthetase binds specifically with its promoter DNA and forms a dimer of dimers. On the other hand the two mutant variants were unable to bind with the DNA. Conclusion: In this study it was concluded that full length E. coli Alanyl-tRNA synthetase undergoes a conformational change in presence of its promoter DNA leading to formation of higher order structures. However, the exact mechanism behind this binding is currently unknown and beyond the scope of this study.

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