Protein Stabilization in Biosensor Systems

1992 ◽  
pp. 40-55 ◽  
Author(s):  
Timothy D. Gibson ◽  
John R. Woodward
Keyword(s):  
Protein Stabilization

Preparation and Characterization of β-glucosidase Films for Stabilization and Handling in Dry Configurations

2020 ◽  
Vol 21 (8) ◽  
pp. 741-747
Author(s):  
Liguang Zhang ◽  
Yanan Shen ◽  
Wenjing Lu ◽  
Lengqiu Guo ◽  
Min Xiang ◽  
...  
Keyword(s):  
Tensile Strength ◽  
Protein Function ◽  
Protein Stabilization ◽  
Functional Protein ◽  
Elongation At Break ◽  
Gelatin Films ◽  
The Stability ◽  
And Function ◽  
General Method

Background: Although the stability of proteins is of significance to maintain protein function for therapeutical applications, this remains a challenge. Herein, a general method of preserving protein stability and function was developed using gelatin films. Method: Enzymes immobilized onto films composed of gelatin and Ethylene Glycol (EG) were developed to study their ability to stabilize proteins. As a model functional protein, β-glucosidase was selected. The tensile properties, microstructure, and crystallization behavior of the gelatin films were assessed. Result: Our results indicated that film configurations can preserve the activity of β-glucosidase under rigorous conditions (75% relative humidity and 37°C for 47 days). In both control films and films containing 1.8 % β-glucosidase, tensile strength increased with increased EG content, whilst the elongation at break increased initially, then decreased over time. The presence of β-glucosidase had a negligible influence on tensile strength and elongation at break. Scanning electron-microscopy (SEM) revealed that with increasing EG content or decreasing enzyme concentrations, a denser microstructure was observed. Conclusion: In conclusion, the dry film is a promising candidate to maintain protein stabilization and handling. The configuration is convenient and cheap, and thus applicable to protein storage and transportation processes in the future.

Protein Solvent Interaction: Transition of Protein-solvent Interaction Concept from Basic Research into Solvent Manipulation of Chromatography

2018 ◽  
Vol 20 (1) ◽  
pp. 34-39 ◽  
Author(s):  
Tsutomu Arakawa ◽  
Yoshiko Kita
Keyword(s):  
Column Chromatography ◽  
Rapid Development ◽  
Academic Research ◽  
Basic Research ◽  
Protein Stabilization ◽  
Special Issue ◽  
Solvent Interaction ◽  
Protein Formulations ◽  
Solvent Additive ◽  
Downstream Processes

Previously, we have reviewed in this journal (Arakawa, T., Kita, Y., Curr. Protein Pept. Sci., 15, 608-620, 2014) the interaction of arginine with proteins and various applications of this solvent additive in the area of protein formulations and downstream processes. In this special issue, we expand the concept of protein-solvent interaction into the analysis of the effects of solvent additives on various column chromatography, including mixed-mode chromatography. Earlier in our research, we have studied the interactions of such a variety of solvent additives as sugars, salts, amino acids, polymers and organic solvents with a variety of proteins, which resulted in mechanistic understanding on their protein stabilization and precipitation effects, the latter known as Hofmeister series. While such a study was then a pure academic research, rapid development of genetic engineering technologies and resultant biotechnologies made it a valuable knowledge in fully utilizing solvent additives in manipulation of protein solution, including column chromatography.

HIF1A is Overexpressed in Medulloblastoma and its Inhibition Reduces Proliferation and Increases EPAS1 and ATG16L1 Methylation

2018 ◽  
Vol 18 (3) ◽  
pp. 287-294 ◽  
Author(s):  
Gustavo Alencastro Veiga Cruzeiro ◽  
Maristella Bergamo dos Reis ◽  
Vanessa Silva Silveira ◽  
Regia Caroline Peixoto Lira ◽  
Carlos Gilberto Carlotti Jr ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cell Line ◽  
Cellular Proliferation ◽  
Mrna Level ◽  
Relevant Information ◽  
Protein Stabilization ◽  
Mrna Levels ◽  
Medulloblastoma Cell Line ◽  
Pediatric Medulloblastoma ◽  
Fresh Frozen

Background: Genetic and epigenetic modifications are closely related to tumor initiation and progression and can provide guidance for understanding tumor functioning, potentially leading to the discovery of new therapies. Studies have associated hypoxia-related genes to tumor progression and chemo/radioresistance in brain tumors. Information on the expression profile of hypoxiarelated genes in pediatric medulloblastoma, although scarce, may reveal relevant information that could support treatment decisions. Objective: Our study focused on evaluation the of CA9, CA12, HIF1A, EPAS1, SCL2A1 and VEGF genes in 41 pediatric fresh-frozen medulloblastoma sample. Additionally, we analyzed the effect of hypoxia and normoxia in the pediatric medulloblastoma cell-line UW402. Furthermore, we assessed the effects of HIF1A knockdown in cell-proliferation and methylation levels of genes related to hypoxia, apoptosis and autophagy. Method: qPCR was performed to evaluate mRNA levels, and Western blot to confirm HIF1A silencing in both patient samples and cell line. Pyrosequencing was performed to asses the methylation levels after HIF1A knockdown in the UW402 cell line. Results: A higher HIF1A mRNA level was observed in MB patients when compared to the cerebellum (non-tumor match). In UW402 MB cell-line, chemically induced hypoxic resulted in an increase of mRNA levels of HIF1A, VEGF, SCL2A1 and CA9 genes. Additionally, HIF1A knockdown induced a decrease in the expression of hypoxia related genes and a decrease of 30% in cell proliferation was also observed. Also, a significant increase in the methylation of ATG16L1 promoter and decrease in the methylation of EPAS1 promoter were observed after HIF1A knockdown. Conclusion: HIF1A knockdown in medulloblastoma cells lead to decreased cellular proliferation, suggesting that HIF1A can be a potential therapeutic target to be explored in the medulloblastoma. However, the mechanisms behind HIF1A protein stabilization and function are very complex and more data need to be generated to potentially use HIF1A as a therapeutical target.

Mechanism of Trehalose-Induced Protein Stabilization from Neutron Scattering and Modeling

2019 ◽  
Vol 123 (17) ◽  
pp. 3679-3687 ◽  
Author(s):  
Christoffer Olsson ◽  
Samuel Genheden ◽  
Victoria García Sakai ◽  
Jan Swenson
Keyword(s):  
Neutron Scattering ◽  
Protein Stabilization

X-ray crystallographic validation of structure predictions used in computational design for protein stabilization

2015 ◽  
Vol 83 (5) ◽  
pp. 940-951 ◽  
Author(s):  
Robert J. Floor ◽  
Hein J. Wijma ◽  
Peter A. Jekel ◽  
Anke C. Terwisscha van Scheltinga ◽  
Bauke W. Dijkstra ◽  
...  
Keyword(s):  
Computational Design ◽  
Protein Stabilization ◽  
X Ray

From the discovery to molecular understanding of cellular iron-sulfur protein biogenesis

2020 ◽  
Vol 401 (6-7) ◽  
pp. 855-876 ◽  
Author(s):  
Roland Lill
Keyword(s):  
De Novo ◽  
Current Knowledge ◽  
Protein Stabilization ◽  
S Protein ◽  
Cellular Iron ◽  
Iron Sulfur ◽  
Protein Biogenesis ◽  
Sulfur Protein ◽  
Initial Discovery ◽  
S Proteins

AbstractProtein cofactors often are the business ends of proteins, and are either synthesized inside cells or are taken up from the nutrition. A cofactor that strictly needs to be synthesized by cells is the iron-sulfur (Fe/S) cluster. This evolutionary ancient compound performs numerous biochemical functions including electron transfer, catalysis, sulfur mobilization, regulation and protein stabilization. Since the discovery of eukaryotic Fe/S protein biogenesis two decades ago, more than 30 biogenesis factors have been identified in mitochondria and cytosol. They support the synthesis, trafficking and target-specific insertion of Fe/S clusters. In this review, I first summarize what led to the initial discovery of Fe/S protein biogenesis in yeast. I then discuss the function and localization of Fe/S proteins in (non-green) eukaryotes. The major part of the review provides a detailed synopsis of the three major steps of mitochondrial Fe/S protein biogenesis, i.e. the de novo synthesis of a [2Fe-2S] cluster on a scaffold protein, the Hsp70 chaperone-mediated transfer of the cluster and integration into [2Fe-2S] recipient apoproteins, and the reductive fusion of [2Fe-2S] to [4Fe-4S] clusters and their subsequent assembly into target apoproteins. Finally, I summarize the current knowledge of the mechanisms underlying the maturation of cytosolic and nuclear Fe/S proteins.

scholarly journals A novel Keap1 inhibitor iKeap1 activates Nrf2 signaling and ameliorates hydrogen peroxide-induced oxidative injury and apoptosis in osteoblasts

2021 ◽  
Vol 12 (7) ◽  
Author(s):  
Yue-huan Zheng ◽  
Jian-jun Yang ◽  
Pei-jun Tang ◽  
Yuan Zhu ◽  
Zhe Chen ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Nuclear Translocation ◽  
Oxidative Injury ◽  
Potential Effect ◽  
Protein Stabilization ◽  
Human Osteoblasts ◽  
Nrf2 Knockout ◽  
Anti Oxidant ◽  
Species Production ◽  
Nrf2 Protein

AbstractAn ultra-large structure-based virtual screening has discovered iKeap1 as a direct Keap1 inhibitor that can efficiently activate Nrf2 signaling. We here tested its potential effect against hydrogen peroxide (H2O2)-induced oxidative injury in osteoblasts. In primary murine and human osteoblasts, iKeap1 robustly activated Nrf2 signaling at micromole concentrations. iKeap1 disrupted Keap1-Nrf2 association, causing Nrf2 protein stabilization, cytosol accumulation and nuclear translocation in murine and human osteoblasts. The anti-oxidant response elements (ARE) activity and transcription of Nrf2-ARE-dependent genes (including HO1, NQO1 and GCLC) were increased as well. Significantly, iKeap1 pretreatment largely ameliorated H2O2-induced reactive oxygen species production, lipid peroxidation and DNA damage as well as cell apoptosis and programmed necrosis in osteoblasts. Moreover, dexamethasone- and nicotine-induced oxidative injury and apoptosis were alleviated by iKeap1. Importantly, Nrf2 shRNA or CRISPR/Cas9-induced Nrf2 knockout completely abolished iKeap1-induced osteoblast cytoprotection against H2O2. Conversely, CRISPR/Cas9-induced Keap1 knockout induced Nrf2 cascade activation and mimicked iKeap1-induced cytoprotective actions in murine osteoblasts. iKeap1 was ineffective against H2O2 in the Keap1-knockout murine osteoblasts. Collectively, iKeap1 activated Nrf2 signaling cascade to inhibit H2O2-induced oxidative injury and death of osteoblasts.

scholarly journals Membrane Protein Stabilization Strategies for Structural and Functional Studies

Membranes ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 155
Author(s):  
Ekaitz Errasti-Murugarren ◽  
Paola Bartoccioni ◽  
Manuel Palacín
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Lipid Membrane ◽  
Protein Stabilization ◽  
New Drugs ◽  
Cell Physiology ◽  
Protein Preparation ◽  
Functional Studies ◽  
Membrane Protein Stability ◽  
Druggable Targets

Accounting for nearly two-thirds of known druggable targets, membrane proteins are highly relevant for cell physiology and pharmacology. In this regard, the structural determination of pharmacologically relevant targets would facilitate the intelligent design of new drugs. The structural biology of membrane proteins is a field experiencing significant growth as a result of the development of new strategies for structure determination. However, membrane protein preparation for structural studies continues to be a limiting step in many cases due to the inherent instability of these molecules in non-native membrane environments. This review describes the approaches that have been developed to improve membrane protein stability. Membrane protein mutagenesis, detergent selection, lipid membrane mimics, antibodies, and ligands are described in this review as approaches to facilitate the production of purified and stable membrane proteins of interest for structural and functional studies.

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