Use of a special Brazilian red-light emitting railroad worm Luciferase in bioassays of NEK7 protein Kinase and Creatine Kinase

Author(s)

Perez, Arina Marina ; Aquino, Bruno ; Viviani, Vadim ; Kobarg, Jorg

• Document type: Journal article
• Source: BMC BIOCHEMISTRY; v. 18, JUL 19 2017.
• Web of Science Citations: 0

Abstract

Background: Luciferases, enzymes that catalyze bioluminescent reactions in different organisms, have been extensively used for bioanalytical purposes. The most well studied bioluminescent system is that of firefly and other beetles, which depends on a luciferase, a benzothiazolic luciferin and ATP, and it is being widely used as a bioanalytical reagent to quantify ATP. Protein kinases are proteins that modify other proteins by transferring phosphate groups from a nucleoside triphosphate, usually ATP. Methods: Here, we used a red-light emitting luciferase from Phrixotrix hirtus railroad worm to determine the activity of kinases in a coupled assay, based on luminescence that is generated when luciferase is in the presence of its substrate, the luciferin, and ATP. Results: In this work we used, after several optimization reactions, creatine kinase isoforms as well as NEK7 protein kinase in the absence or presence of ATP analogous inhibitors to validate this new luminescence method. Conclusion: With this new approach we validated a luminescence method to quantify kinase activity, with different substrates and inhibition screening tests, using a novel red-light emitting luciferase as a reporter enzyme. (AU)

• FAPESP's process: 11/20086-1 - Confirmation and functional analysis of the interaction of new proteins that interact with the cytoplasmic region of CD30 the marker protein for Hodgin lymphoma
• Grantee: Bruno Aquino
• Support Opportunities: Scholarships in Brazil - Doctorate
• FAPESP's process: 13/09594-0 - Analysis of coleopteran lanterns, fat body and Malpighian tubules transcriptomes, and its relationship with the origins of bioluminescence in Coleoptera and Diptera
• Grantee: Vadim Viviani
• Support Opportunities: Regular Research Grants
• FAPESP's process: 14/21700-3 - Functional characterization of mice and their cells in which the genes encoding the regulatory proteins Ki-1/57 and CGI-55 have been knocked out
• Grantee: Jörg Kobarg
• Support Opportunities: Regular Research Grants
• FAPESP's process: 12/22265-3 - Study of the physiological role of Nek7 protein in cell cycle and tumorigenesis
• Grantee: Arina Marina Perez
• Support Opportunities: Scholarships in Brazil - Post-Doctoral