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

Autor(es)

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

• Tipo de documento: Artigo Científico
• Fonte: BMC BIOCHEMISTRY; v. 18, JUL 19 2017.
• Citações Web of Science: 0

Resumo

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)

• Processo FAPESP: 11/20086-1 - Confirmação e análise funcional da interação de novas proteínas que interagem com a região intracelular do marcador de linfoma de Hodgin CD30
• Beneficiário: Bruno Aquino
• Linha de fomento: Bolsas no Brasil - Doutorado
• Processo FAPESP: 13/09594-0 - Análise dos transcriptomas das laternas, corpo gorduroso e túbulos de Malpighi de coleópteros, e suas relações com as origens da bioluminescência em Coleoptera e Diptera
• Beneficiário: Vadim Viviani
• Linha de fomento: Auxílio à Pesquisa - Regular
• Processo FAPESP: 14/21700-3 - Caracterização funcional de camundongos e células nocaute para os genes que codificam as proteínas regulatórias Ki-1/57 e CGI-55
• Beneficiário: Jörg Kobarg
• Linha de fomento: Auxílio à Pesquisa - Regular
• Processo FAPESP: 12/22265-3 - Estudo do Papel fisiológico da proteína Nek7 , no ciclo celular e na tumorigênese
• Beneficiário: Arina Marina Perez
• Linha de fomento: Bolsas no Brasil - Pós-Doutorado