scholarly journals A sensitive, accurate, and high-throughput gluco-oligosaccharide oxidase based HRP colorimetric method for lytic polysaccharide monooxygenase activity assay

2021 ◽  
Author(s):  
Gang Liu
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Direct Method ◽  
Total Concentration ◽  
Colorimetric Method ◽  
Industrial Applications ◽  
Assay Method ◽  
Activity Assay ◽  
Lytic Polysaccharide Monooxygenase ◽  
Monooxygenase Activity

Abstract Background The AA9 (auxiliary activities) family of lytic polysaccharide monooxygenases (AA9 LPMOs) are ubiquitous and diverse group of enzymes amongst the fungal kingdom. They catalyze the oxidative cleavage of glycosidic bonds in lignocellulose and exhibit great potential for secondary biorefinery applications. Screening of AA9 LPMOs for desirable properties is crucial for biorefinery industrial applications. However, robust, high-throughput and direct method for AA9 LPMO activity assay, which is prerequisite for screening of LPMOs with excellent properties, is still lacking. Here, we have described a gluco-oligosaccharide oxidase (GOOX) based horseradish peroxidase (HRP) colorimetric method for AA9 LPMO activity assay. Results We cloned and expressed a GOOX gene from Sarocladium strictum in Trichoderma reesei, purified the recombinant SsGOOX, validated its properties, and set up a SsGOOX based HRP colorimetric method for cellobiose concentration assay. Then we expressed two AA9 LPMOs from Thielavia terrestris, TtAA9F and TtAA9G in T. reesei, purified the recombinant proteins, and analyzed their product profiles and regioselectivity towards phosphoric acid swollen cellulose (PASC). TtAA9F was characterized as a C1 type (class 1) LPMO, while TtAA9G was characterized as a C4 type (class 2) LPMO. Finally, the SsGOOX based HRP colorimetric method was used to quantify the total concentration of reducing lytic products from LPMO reaction, and consequently, the activities of both C1 and C4 types of LPMOs were analyzed. These LPMOs could be effectively analyzed with limits of detection (LoDs) lower than 30 nmol/L, and standard curves between A515 and LPMO concentrations with determination coefficients greater than 0.994 were obtained. Conclusions A novel, sensitive and accurate assay method that directly targets the main activity of both C1 and C4 type of AA9 LPMOs was established. This method is easy to use and could be performed on a microtiter plate ready for high-throughput screening of AA9 LPMOs with high properties.

scholarly journals A Direct Fluorescent Activity Assay for Glycosyltransferases Enables Convenient High‐Throughput Screening: Application to O ‐GlcNAc Transferase

2020 ◽  
Vol 59 (24) ◽  
pp. 9601-9609 ◽  
Author(s):  
Matthew G. Alteen ◽  
Christina Gros ◽  
Richard W. Meek ◽  
David A. Cardoso ◽  
Jil A. Busmann ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Activity Assay ◽  
Glcnac Transferase

A robust and quantitative assay platform for multiplexed, high throughput screening of protein kinase inhibitors

2016 ◽  
Vol 52 (81) ◽  
pp. 12112-12115 ◽  
Author(s):  
Jieon Lee ◽  
Il-Soo Park ◽  
Ginam Park ◽  
Kyukwang Cho ◽  
Hee-Sung Park ◽  
...  
Keyword(s):  
Graphene Oxide ◽  
Protein Kinase ◽  
High Throughput ◽  
High Throughput Screening ◽  
Kinase Activity ◽  
Kinase Inhibitors ◽  
Protein Kinase Inhibitors ◽  
Protein Kinase Activity ◽  
Activity Assay ◽  
Kinase Activity Assay

We present a new platform for multiplexed protein kinase activity assay using TiO2decorated graphene oxide (GO), which is applicable to high throughput inhibitor screening.

scholarly journals High-Throughput Screening Method of Inhibitors that Block the Interaction between 2 Helical Regions of HIV-1 gp41

2005 ◽  
Vol 10 (1) ◽  
pp. 13-19 ◽  
Author(s):  
Bong-Suk Jin ◽  
Won-Kyu Lee ◽  
Kwangseog Ahn ◽  
Myung Kyu Lee ◽  
Yeon Gyu Yu
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Screening Method ◽  
Assay Method ◽  
Enzyme Linked Immunosorbent Assay ◽  
Compound Library ◽  
Elisa Method ◽  
Helical Bundle ◽  
Fusion Inhibitors ◽  
Hiv 1

The HIV-1 envelope glycoprotein transmembrane subunit, gp41, mediates the fusion of viral and target cell membranes. The 2 helical regions in the ectodomain of gp41, the N-helix and the C-helix, form a helical bundle complex that has been suggested as a fusion-active conformation. Previously, an enzyme-linked immunosorbent assay (ELISA) method had been established to measure the interaction of 2 helical regions of gp41. In this study, the ELISA method was modified to apply high-throughput screening (HTS) of an organic compound library. A few compounds had been identified to prevent the interaction between 2 helical regions of gp41, and they were further shown to inhibit the gp41-mediated viral infection. In addition, they specifically quenched the fluorescence of tryptophan in the N-helix region, indicating that these compounds bound to the N-helix rather than the C-helix of gp41. These results suggested that this assay method targeting gp41 could be used for HTS of HIV fusion inhibitors. ( Journal of Biomolecular Screening 2005:13-19)

scholarly journals High-throughput direct screening of restriction endonuclease using microfluidic fluorescence-activated drop sorter based on SOS response in E. coli

2022 ◽  
Author(s):  
Yizhe Zhang ◽  
Jeremy J Agresti ◽  
Yu Zheng ◽  
David A Weitz
Keyword(s):  
Restriction Endonuclease ◽  
High Throughput ◽  
High Throughput Screening ◽  
Large Scale ◽  
Screening Method ◽  
Sos Response ◽  
Industrial Applications ◽  
Ease Of Use ◽  
Isolation And Characterization ◽  
New Strategy

A restriction endonuclease (RE) is an enzyme that can recognize a specific DNA sequence and cleave that DNA into fragments with double-stranded breaks. This sequence-specific cleaving ability and its ease of use have made REs commonly used tools in molecular biology since their first isolation and characterization in 1970s. While artificial REs still face many challenges in large-scale synthesis and precise activity control for practical use, searching for new REs in natural samples remains a viable route for expanding the RE pool for fundamental research and industrial applications. In this paper, we propose a new strategy to search for REs in an efficient fashion. Briefly, we construct a host bacterial cell to link the RE genotype to the phenotype of β-galactosidase expression based on the bacterial SOS response, and use a high-throughput microfluidic platform to isolate, detect and sort the REs. We employ this strategy to screen for the XbaI gene from constructed libraries of varied sizes. In single round of sorting, a 30-fold target enrichment was obtained within 1 h. The direct screening approach we propose shows potential for efficient search of desirable REs in natural samples compared to the conventional RE-screening method, and is amenable to being adapted to high-throughput screening of other genotoxic targets.

scholarly journals Development and Validation of a High-Throughput Intrinsic ATPase Activity Assay for the Discovery of MEKK2 Inhibitors

2012 ◽  
Vol 18 (4) ◽  
pp. 388-399 ◽  
Author(s):  
Syed Ahmad ◽  
Mark A. Hughes ◽  
Gary L. Johnson ◽  
John E. Scott
Keyword(s):  
Atpase Activity ◽  
High Throughput ◽  
High Throughput Screening ◽  
Selective Inhibition ◽  
Mass Spectrometry Analysis ◽  
Activity Assay ◽  
Spectrometry Analysis ◽  
Compound Libraries ◽  
Atpase Assay

The kinase MEKK2 (MAP3K2) has recently been implicated in tumor growth and metastasis. Thus, selective inhibition of MEKK2 may be a novel strategy for cancer therapy. To identify inhibitors of MEKK2 kinase activity, we have developed a novel activity assay for MEKK2 based on the discovery that recombinant purified MEKK2 has intrinsic ATPase activity. This MEKK2 ATPase assay was validated for enzyme identity and enzymatic purity by multiple methods including mass spectrometry analysis, testing different sources of MEKK2 and comparing ATPase assay IC50 data for multiple inhibitors to literature values and to IC50 data generated using MEKK2 binding and transphosphorylation assays. Taken together, these data indicated that genuine MEKK2 activity was being measured in this assay and no other ATPases contributed to the signal. A miniaturized version of the assay was validated for high-throughput screening, and compound libraries were screened. The screening hits generated comparable potencies in the MEKK2 intrinsic ATPase, binding, and transphosphorylation assays. We identified a novel MEKK2 inhibitor and confirmed that crizotinib and bosutinib are potent in vitro inhibitors of MEKK2 activity with IC50 values of <100 nM. Thus, this assay has utility for the discovery of small-molecule inhibitors of MEKK2 activity.

Homogeneous single-label tyrosine kinase activity assay for high throughput screening

2015 ◽  
Vol 897 ◽  
pp. 96-101 ◽  
Author(s):  
Natalia Tong-Ochoa ◽  
Kari Kopra ◽  
Markku Syrjänpää ◽  
Nicolas Legrand ◽  
Harri Härmä
Keyword(s):  
Tyrosine Kinase ◽  
High Throughput ◽  
High Throughput Screening ◽  
Kinase Activity ◽  
Activity Assay ◽  
Tyrosine Kinase Activity ◽  
Kinase Activity Assay

scholarly journals High-Throughput Screen for Escherichia coli Heat Shock Protein 70 (Hsp70/DnaK)

2010 ◽  
Vol 15 (10) ◽  
pp. 1211-1219 ◽  
Author(s):  
Yoshinari Miyata ◽  
Lyra Chang ◽  
Anthony Bainor ◽  
Thomas J. Mcquade ◽  
Christopher P. Walczak ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
High Throughput ◽  
Heat Shock Protein 70 ◽  
High Throughput Screening ◽  
Colorimetric Method ◽  
Hsp70 Family ◽  
Assay Performance ◽  
Phosphate Detection

Members of the heat shock protein 70 (Hsp70) family of molecular chaperones are emerging as potential therapeutic targets. Their ATPase activity has classically been measured using colorimetric phosphate detection reagents, such as quinaldine red (QR). Although such assays are suitable for 96-well plate formats, they typically lose sensitivity when attempted in lower volume due to path length and meniscus effects. These limitations and Hsp70’s weak enzymatic activity have combined to create significant challenges in high-throughput screening. To overcome these difficulties, the authors have adopted an energy transfer strategy that was originally reported by Zuck et al. ( Anal Biochem 2005;342:254-259). Briefly, white 384-well plates emit fluorescence when irradiated at 430 nm. In turn, this intrinsic fluorescence can be quenched by energy transfer with the QR-based chromophore. Using this more sensitive approach, the authors tested 55,400 compounds against DnaK, a prokaryotic member of the Hsp70 family. The assay performance was good (Z′ ~0.6, coefficient of variation ~8%), and at least one promising new inhibitor was identified. In secondary assays, this compound specifically blocked stimulation of DnaK by its co-chaperone, DnaJ. Thus, this simple and inexpensive adaptation of a colorimetric method might be suitable for screening against Hsp70 family members.

Development of a High-Throughput Assay to Identify Inhibitors of ENPP1

2021 ◽  
pp. 247255522098232
Author(s):  
Meera Kumar ◽  
Robert G. Lowery
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Type I Interferon ◽  
Direct Detection ◽  
Innate Immune ◽  
Assay Method ◽  
Type I ◽  
Immune Response To Cancer ◽  
Z Value ◽  
High Throughput Assay

The innate immune response to cancer is initiated by cytosolic DNA, where it binds to cGAS and triggers type I interferon (IFN) expression via the STING receptor, leading to activation of tumor-specific T cells. Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) has been identified as the primary enzyme responsible for degrading cGAMP, and therefore it is under intense investigation as a therapeutic target for cancer immunotherapy. ENPP1 hydrolyzes cGAMP to produce AMP and GMP, and hydrolyzes ATP and other nucleotides to monophosphates and pyrophosphate. We developed a robust, high-throughput screening (HTS)-compatible enzymatic assay method for ENPP1 using the Transcreener AMP2/GMP2 Assay, a competitive fluorescence polarization (FP) immunoassay that enables direct detection of AMP and GMP in a homogenous format. The monoclonal antibody used in the Transcreener AMP2/GMP2 Assay showed more than 104-fold selectivity for AMP and GMP versus cGAMP, and 3000-fold selectivity for AMP over ATP, indicating that the assay can be used for detection at initial velocity with either substrate. A working concentration of 100 pM ENPP1 was determined as optimal with a 60 min reaction period, enabling screening with very low quantities of enzyme. A Z′ value of 0.72 was determined using ATP as substrate, indicating a high-quality assay. Consistent with previous studies, we found that ENPP1 preferred ATP as a substrate when compared with other nucleotides like GTP, ADP, and GDP. ENPP1 showed a 20-fold selectivity for 2′3′cGAMP compared with 2′3′c-diGMP and showed no activity with 3′3′c-diAMP. The Transcreener AMP2/GMP2 Assay should prove to be a valuable tool for the discovery of ENPP1 lead molecules.

scholarly journals Enzyme Functional Screening, Discovery and Engineering; Automation, Metagenomics and High-throughput Approaches

2020 ◽  
pp. 1-12
Author(s):  
Sikander Ali ◽  
Syed Shahid Hussain
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Industrial Applications ◽  
Enzyme Engineering ◽  
Functional Screening ◽  
Functional Metagenomics ◽  
Immense Potential

Concerned with the construction and design of novel biocatalysts, the enzyme engineering served to overcome the limitations of native enzymes, in order to create biocatalysts with tailored functions, to facilitate industrial applications. The enzymes, being recognized by screening and discovery workflows and further tailored by engineering platforms, are of immense potential as improved biocatalysts. Functional metagenomics is a powerful tool to identify novel enzymes followed by the construction of metagenome-based enzyme libraries. And the subsequent screening of these enzyme libraries is in turn facilitated by ultra-high-throughput-based, for example FACS or microfluidics, enzyme engineering technologies. Relies on the compartmentalization of reaction components, in order to detect and measure assay signal within the reaction compartments, the enzyme engineering platforms are designed which include cell-as-compartment platforms, droplet-based platforms and micro-chamber-based platforms. The metagenomics approach and high-throughput screening by these three prime enzyme engineer platforms are the focus of this review.

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