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Nucleoside Triphosphate Hydrolases Assay in Toxoplasm gondii and Neospora caninum for High-Throughput Screening using a Robot Arm
* These authors contributed equally
In This Article
Summary
Toxoplasma gondii and Neospora caninum infections are found in humans and animals and lead to serious health issues. The two parasites share similar nucleoside triphosphate hydrolases and play important roles in propagation and survival. We established a high-standard assay of the enzymes requiring robot arm usage.
Abstract
Protozoan parasites infect humans and many warm-blooded animals. Toxoplasma gondii, a major protozoan parasite, is commonly found in HIV-positive patients, organ transplant recipients and pregnant women, resulting in the severe health condition, Toxoplasmosis. Another major protozoan, Neospora caninum, which bears many similarities to Toxoplasma gondii, causes serious diseases in animals, as does Encephalomyelitis and Myositis-Polyradiculitis in dogs and cows, resulting in stillborn calves. All these exhibited similar nucleoside triphosphate hydrolases (NTPase). Neospora caninum has a NcNTPase, while Toxoplasma gondii has a TgNTPase-I. The enzymes are thought to play crucial roles in propagation and survival. In order to establish compounds and/or extracts preventing protozoan infection, we targeted these enzymes for drug discovery. The next step was to establish a novel, highly sensitive, and highly accurate assay by combining a conventional biochemical enzyme assay with a fluorescent assay to determine ADP content. We also validated that the novel assay fulfills the criteria to carry out high-throughput screening (HTS) in the two protozoan enzymes. We performed HTS, identified 19 compounds and six extracts from two synthetic compound libraries and an extract library derived from marine bacteria, respectively. In this study, a detailed explanation has been introduced on how to carry out HTS, including information about the preparation of reagents, devices, robot arm, etc.
Introduction
Robotics have been established as sophisticated and powerful tools for achieving significant breakthroughs in various fields beyond industry and fabrication engineering, such as biochemistry, molecular biology, and clinical research, and notably HTS1,2,3. Toxoplasma gondii is a major parasite and a single-cell parasitic eukaryote4 that causes serious health issues in humans5 and many homeothermic animals4, resulting in infections leading to Toxoplasmosis, a particularly severe condition in AIDS p....
Protocol
1. Expression and purification of recombinant TgNTPase-I and NcNTPase
- Prepare the expression plasmid and introduce it to the E. coli. strain BL21.
​NOTE: Detailed information on constructs and procedures is shown in a previous report14. In this study, both TgNTPase-I and NcNTPase constitutively active mutants were kindly gifted by Prof. Asai and Prof. Harada.
2. Preparation and placement of biofluorescent r.......
Representative Results
A principle of the assay is summarized in Supplementary Figure 1 and based on a previous report12,18. The assay was designed in a 384-well format, as shown in Figure 1. The far-right and left lines were avoided on the plate. The two lines next to the far left and right lines were then used as negative control and positive control with or without the enzyme, respectively (n = 16). This allowed for the 320 compounds on.......
Discussion
We succeeded in establishing a novel high-dynamic range and -accuracy assay with a combination of a classical enzyme assay and a fluorescent assay for ADP, which is the end product through ATPase, including Tg and Nc ATPase22. In order to carry out HTS, it is important that the assay has better values of S/B, S/N, and Z' factor than a classical enzyme assay15,22. Additionally, omitting the step of stopping the enzyme reaction with an acid .......
Acknowledgements
This work was partly supported by the Platform for Drug Discovery, Informatics and Structural Life Science, a Grant-in-Aid for Scientific Research (C) from Japan Society for Promotion of Science (JSPS-21K06566). The authors sincerely thank Asai (Keio University School of medicine) and Harada (Kyoto Institute of Technology) and Stephen Stratton for gifting recombinant two NTPase active mutants and his contribution in the preparation of this manuscript, respectively.
....Materials
| Name | Company | Catalog Number | Comments |
| 12 stage-workstation EDR-384 SX | Biotec Co., Ltd. | EDR-384SX | Robot arm Pippeting system |
| 384 well tips | Biotech Co., Ltd. | Custom made | |
| ADP-hexokinase | Asahi Kasei Pharma Co., Ltd. | T-92 | |
| ATP | Oriental Yeast, Co, Ltd. | 45140000 | |
| BSA | Wako Pure Chemical Industries, Ltd. | 011-15144 | |
| Diaphorase-I | Unitika Ltd. | Di-1 | |
| DMSO | Nacalai Tesch, Inc. | 13406-55 | |
| G6P dehydrogenase | Oriental Yeast, Co, Ltd. | 306-50143 | |
| Glucose | Wako Pure Chemical Industries, Ltd. | 049-31165 | |
| Greiner 384 well micro-plate non-binding shallow well Black | #784900 | ||
| HEPES | Wako Pure Chemical Industries, Ltd. | 342-01375 | |
| Mg(CH3COO)2 | Wako Pure Chemical Industries, Ltd. | 130-00095 | |
| NADP | Oriental Yeast, Co, Ltd. | 44332000 | |
| N-ethylmaleimide | Wako Pure Chemical Industries, Ltd. | 056-02062 | |
| PHERAstar FS | BMG LABTECH JAPAN L.t.d. | PHERAstar FS | Multimode microplate reader |
| Resazurin | Wako Pure Chemical Industries, Ltd. | 191-07581 | |
| Seahorse Labware 384 Well Low profile reservoirs | S30022 25/CS | ||
| TrisHCl | Wako Pure Chemical Industries, Ltd. | W01COBQE-4120 | |
| Triton X-100 | Nacalai Tesch, Inc. | 35501-02 |
References
- Bianca, C. B., et al. A robotic platform to screen aqueous two-phase systems for overcoming inhibition in enzymatic reactions. Bioresource Technology. 280, 37-50 (2019).
- Aliaksei, V., Jan, D. B.
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