Improved protocol for Bst polymerase and reverse transcriptase production and application to a point-of-care diagnostics system

Author(s):Show less -

de Souza, Lucas Rodrigo ; Poly da Silva, Italo Esposti ; Celis-Silva, Gabriele ; Raddatz, Bruna Winkert ; Imamura, Louise Matie ; Kim, Edson Yu Sin ; Valderrama, Gabriel Vieira ; Riedi, Halanna de Paula ; Rogal Jr, Sergio Renato ; Machado de Almeida, Bernardo Montesanti ; Mazega Figueredo, Marcus Vinicius ; Bengtson, Mario Henrique ; Massirer, Katlin Brauer

• Document type: Journal article
• Source: Experimental Biology and Medicine; v. 248, n. 19, p. 13-pg., 2023-12-13.

Abstract

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has raised awareness in the scientific community about the importance of being prepared for sanitary emergencies. Many measures implemented during the COVID pandemic are now being expanded to other applications. In the field of molecular and immunological diagnostics, the need to massively test the population worldwide resulted in the application of a variety of methods to detect viral infection. Besides gold standard reverse transcription quantitative polymerase chain reaction (RT-qPCR), the use of reverse transcription loop-mediated isothermal amplification (RT-LAMP) arose as an alternative and sensitive method to amplify and detect viral genetic material. We have used openly available protocols and have improved the protein production of RT-LAMP enzymes Bst polymerase and HIV-reverse transcriptase. To optimize enzyme production, we tested different protein tags, and we shortened the protein purification protocol, resulting in reduced processing time and handling of the enzymes and, thus, preserved the protein activity with high purity. The enzymes showed significant stability at 4 degrees C and 25 degrees C, over 60 days, and were highly reliable when used as a one-step RT-LAMP reaction in a portable point-of-care device with clinical samples. The enzymes and the reaction setup can be further expanded to detect other infectious diseases agents. (AU)

• FAPESP's process: 20/16094-8 - Structural charaterization of RNA binding proteins and fragment-based screening by Liquid Chromatography and mass spectrometry
• Grantee: Lucas Rodrigo de Souza
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• FAPESP's process: 20/02006-0 - Molecular and structural characterization of RNA binding proteins acting as potential disease targets in cancer and neurology
• Grantee: Katlin Brauer Massirer
• Support Opportunities: Research Grants - Research Partnership for Technological Innovation - PITE
• FAPESP's process: 14/50897-0 - INCT 2014: Open-acess Medicinal Chemistry Centre (OpenMedChem)
• Grantee: Katlin Brauer Massirer
• Support Opportunities: Research Projects - Thematic Grants
• FAPESP's process: 19/11320-2 - Cellular investigation of protein kinases involved with splicing, by minigene system
• Grantee: Gabriel Vieira Valderrama
• Support Opportunities: Scholarships in Brazil - Scientific Initiation