scholarly journals Label-Free Electrochemical Diagnosis of Viral Antigens with Genetically Engineered Fusion Protein

Sensors ◽  
2012 ◽  
Vol 12 (8) ◽  
pp. 10097-10108 ◽  
Author(s):  
Nam Su Heo ◽  
Shun Zheng ◽  
MinHo Yang ◽  
Seok Jae Lee ◽  
Sang Yup Lee ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Genetically Engineered ◽  
Label Free ◽  
Viral Antigens

Development of label-free optical diagnosis for sensitive detection of influenza virus with genetically engineered fusion protein

Talanta ◽  
2012 ◽  
Vol 89 ◽  
pp. 246-252 ◽  
Author(s):  
Tae Jung Park ◽  
Seok Jae Lee ◽  
Do-Kyun Kim ◽  
Nam Su Heo ◽  
Jung Youn Park ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Fusion Protein ◽  
Genetically Engineered ◽  
Sensitive Detection ◽  
Label Free ◽  
Optical Diagnosis

A Genetically Engineered Fusion Protein with Horseradish Peroxidase as a Marker Enzyme for Use in Competitive Immunoassays

2001 ◽  
Vol 73 (6) ◽  
pp. 1134-1139 ◽  
Author(s):  
Vitaly Grigorenko ◽  
Irina Andreeva ◽  
Torsten Börchers ◽  
Friedrich Spener ◽  
Alexey Egorov
Keyword(s):  
Horseradish Peroxidase ◽  
Fusion Protein ◽  
Genetically Engineered ◽  
Marker Enzyme

Cytotoxicity of Genetically Engineered Fusion Protein with CD154 Peptide Mimetic (OmpC-CD154p) on B-NHL Cell Lines.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4525-4525
Author(s):  
Bernardo Martinez-Miguel ◽  
Melisa A. Martinez-Paniagua ◽  
Sara Huerta-Yepez ◽  
Rogelio Hernandez-Pando ◽  
Cesar R. Gonzalez-Bonilla ◽  
...  
Keyword(s):  
Cell Death ◽  
B Cells ◽  
Fusion Protein ◽  
B Cell ◽  
Tumor Cells ◽  
Cell Lines ◽  
B Cell Lymphoma ◽  
Genetically Engineered ◽  
B Cell Malignancies ◽  
Peptide Mimetic

Abstract The interaction between CD40, a member of the tumor necrosis factor super family, and its ligand CD154 is essential for the development of humoral and cellular immune responses. Selective inhibition or activation of this pathway forms the basis for the development of new therapeutics against immunologically-based diseases and malignancies. CD40 is expressed primarily on dendritic cells, macrophages and B cells. Engagement of CD40-CD154 induces activation and proliferation of B lymphocytes and triggers apoptosis of carcinoma and B lymphoma cells. Agonist CD40 antibodies mimic the signal of CD154-CD40 ligation on the surface of many tumors and mediate a direct cytotoxic effect in the absence of immune accessory molecules. CD40 expression is found on nearly all B cell malignancies. Engagement of CD40 in vivo inhibits B cell lymphoma xenografts in immune compromised mice. Several clinical trials have been reported targeting CD40 in cancer patients using recombinant CD154, mAbs and gene therapy, which were well tolerated and resulted in objective tumor responses. In addition to these therapies, CD54 mimetics have been considered with the objective to augment and potentiate the direct cytotoxic anti-tumor activity and for better accessibility to tumor sites. This approach was developed by us and we hypothesized that the genetic engineering of a fusion protein containing a CD154 peptide mimetic may be advantageous in that it may have a better affinity to CD40 on B cell malignancies and trigger cell death and the partner may be a carrier targeting other surface molecules expressed on the malignant cells. This hypothesis was tested by the development of a gene fusion of Salmonella typhi OmpC protein expressing the CD154 Trp140-Ser149 amino acid strand (Vega et al., Immunology2003; 110: 206–216). This OmpC-CD154p fusion protein binds CD40 and triggers the CD40 expressing B cells. In this study, we demonstrate that OmpC-CD154p treatment inhibits cell growth and proliferation of the B-NHL cell lines Raji and Ramos. In addition, significant apoptosis was achieved and the extent of apoptosis was a function of the concentration used and time of incubation. The anti-tumor effect was specific as treatment with OmpC alone had no effect. These findings establish the basis of the development of new fusion proteins with dual specificity (targeting the tumor cells directly or targeting the tumor cells and immune cells). The advantages of this approach over conventional CD40-targeted therapies as well as the mechanism of OmpC-CD154p-induced cell signaling and cell death will be presented.

scholarly journals Overcoming rituximab drug-resistance by the genetically engineered anti-CD20-hIFN-α fusion protein: Direct cytotoxicity and synergy with chemotherapy

2015 ◽  
Vol 47 (5) ◽  
pp. 1735-1748 ◽  
Author(s):  
GABRIEL G. VEGA ◽  
LUZ ARELI FRANCO-CEA ◽  
SARA HUERTA-YEPEZ ◽  
HÉCTOR MAYANI ◽  
SHERIE L. MORRISON ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Fusion Protein ◽  
Genetically Engineered ◽  
Direct Cytotoxicity ◽  
Anti Cd20

High Expression and Preliminary Purification of Human β-Defensin-2 Fusion Protein

2013 ◽  
Vol 781-784 ◽  
pp. 1076-1079
Author(s):  
Hong Tao Wei ◽  
Zhong Wen Lv ◽  
Xue Mei Han ◽  
Guo Li Zhang
Keyword(s):  
Fusion Protein ◽  
Metal Chelate ◽  
Genetically Engineered ◽  
High Expression ◽  
Soluble Form ◽  
Freezing And Thawing ◽  
Preliminary Purification ◽  
Prokaryotic Expression Vector ◽  
Genetically Engineered Bacteria

This study was undertaken to achieve high expression and preliminary purification of human β-defensin-2 fusion protein to lay a solid foundation for production of human β-defensin-2 using genetic engineering. A prokaryotic expression vector for human β-defensin-2 fusion protein was generated using in vitro gene synthesis before transformation into BL21 (l DE3) plysS TrX-B host bacteria. High expression of TrX-A-HBD-2 fusion protein was induced with IPTG in the bacteria exposed to various expression conditions. The fusion protein then underwent preliminary purification. The protein of interest was released from the genetically engineered bacteria after freezing and thawing. The expression of the target protein accounted for 16.12% of the total bacterial proteins. Fractional precipitation with saturated ammonium sulfate and metal chelate affinity chromatography yielded human β-defensin-2 peptide fusion protein, with a relative purity of 80.53%.Human β-defensin-2 fusion protein could be highly expressed in a soluble form, with a relatively high purity

scholarly journals Graphene Biosensor Programming with Genetically Engineered Fusion Protein Monolayers

2016 ◽  
Vol 8 (12) ◽  
pp. 8257-8264 ◽  
Author(s):  
Miika Soikkeli ◽  
Katri Kurppa ◽  
Markku Kainlauri ◽  
Sanna Arpiainen ◽  
Arja Paananen ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Genetically Engineered ◽  
Protein Monolayers

scholarly journals Grating-coupled interferometry reveals binding kinetics and affinities of Ni ions to genetically engineered protein layers

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Hajnalka Jankovics ◽  
Boglarka Kovacs ◽  
Andras Saftics ◽  
Tamas Gerecsei ◽  
Éva Tóth ◽  
...  
Keyword(s):  
Binding Sites ◽  
Kinetic Data ◽  
Specific Binding ◽  
Genetically Engineered ◽  
Binding Kinetics ◽  
Titration Calorimetry ◽  
Affinity Binding ◽  
Label Free ◽  
Low Protein ◽  
Kinetics Of

AbstractReliable measurement of the binding kinetics of low molecular weight analytes to their targets is still a challenging task. Often, the introduction of labels is simply impossible in such measurements, and the application of label-free methods is the only reliable choice. By measuring the binding kinetics of Ni(II) ions to genetically modified flagellin layers, we demonstrate that: (1) Grating-Coupled Interferometry (GCI) is well suited to resolve the binding of ions, even at very low protein immobilization levels; (2) it supplies high quality kinetic data from which the number and strength of available binding sites can be determined, and (3) the rate constants of the binding events can also be obtained with high accuracy. Experiments were performed using a flagellin variant incorporating the C-terminal domain of the nickel-responsive transcription factor NikR. GCI results were compared to affinity data from titration calorimetry. We found that besides the low-affinity binding sites characterized by a micromolar dissociation constant (Kd), tetrameric FliC-NikRC molecules possess high-affinity binding sites with Kd values in the nanomolar range. GCI enabled us to obtain real-time kinetic data for the specific binding of an analyte with molar mass as low as 59 Da, even at signals lower than 1 pg/mm2.

Protein Degradation Profile Reveals Dynamic Nature of 20S CP Small Molecule Stimulation

2020 ◽  
Author(s):  
Rachel A. Coleman ◽  
Tiago Jose Paschoal Sobreira ◽  
Uma Aryal ◽  
Darci Trader
Keyword(s):  
Fusion Protein ◽  
Protein Degradation ◽  
Small Molecule ◽  
Degradation Process ◽  
Cellular Systems ◽  
Label Free ◽  
Dynamic Nature ◽  
Protein Levels ◽  
Gfp Fusion Protein ◽  
The Impact

<p>Small molecules have recently been discovered to stimulate the 20S core particle (CP) of the proteasome to degrade proteins, such as a-synuclein. While these studies have focused on particular proteins that are known 20S CP substrates, it is currently unclear how many or what types of proteins may be affected by enhancing this degradation process. We present here a study that utilizes four 20S CP stimulators to determine how each can affect the degradation of proteins in a biochemical assay with purified proteins, an overexpressed GFP-fusion protein in cells, and the effects of stimulators using label-free quantitative proteomic analysis for a more broad understanding on their impact. The results of these studies highlight that the impact of small molecule stimulators of the 20S CP on protein degradation cannot be easily predicted. While 20S CP stimulators will likely increase the degradation of proteins that have significant disorder, such as a-synuclein and tau, we observed different impacts on the degradation of proteins less than 90% disordered. To gain greater insight into the cellular systems that may be affected by 20S CP stimulators, we analyzed the proteome of HEK-293T cells treated with two of our stimulators, AM-404 or miconazole. These results were then compared to cells treated with a proteasome inhibitor, bortezomib. Our results show that 20S CP stimulators affect a smaller number of proteins as compared to bortezomib. The proteomics analysis corroborates our biochemical and GFP-fusion protein results, confirming that the impact of a 20S CP stimulator on protein degradation is dependent on the stimulator. Taken together, this study reveals the dynamic nature of the 20S CP, as small molecule stimulators can have a variety of mechanisms of action to change protein degradation activity. Our studies show that 20S CP stimulation can lead to a decrease in protein levels, more so those proteins that are significantly disordered, and that small molecule stimulators can potentially be tailored to decrease certain protein types.</p>

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