Cloning, expression and purification of recombinant Thiamine Monophosphate Kinase (ThiL) from clinically relevant enterobacteria

• Grant number: 23/06799-2
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: July 01, 2023
• End date: December 31, 2024
• Field of knowledge: Biological Sciences - Biophysics - Molecular Biophysics
• Principal Investigator: Marcelo Ferreira Marcondes Machado
• Grantee: Isabela Amie Nascimento Matsukuma
• Host Institution: Instituto Nacional de Farmacologia (INFAR). Universidade Federal de São Paulo (UNIFESP). Campus São Paulo. São Paulo , SP, Brazil

Abstract

Due to the emergence of bacteria resistant to current antibiotics, the development of new antibiotics remains one of the highest challenges of modern medicine. The emergence of mechanisms of resistance to the currently most used antimicrobials indicates the importance to develop new therapeutic methods. Thiamine has its biosynthesis present only in prokaryotic organisms, since eukaryotes do not have a cellular machinery capable of carrying out this process, this molecule is described as an important compound for the survival of eukaryotic and prokaryotic cells, due to the actions of different enzymes, for example the thiamine pyrophosphate (TPP) has been described as an important cofactor for metabolic processes. Recent studies demonstrate the potential of thiamine monophosphate kinase (ThiL) as a target for the development of new drugs with specific action in prokaryotic cells. ThiL is responsible for catalyzing the phosphorylation reaction of thiamine monophosphate (TMP) in TPP, an important cofactor for bacterial metabolism. The main objective of this project is to perform the cloning, expression and purification of recombinant ThiL from different enterobacteria, namely: Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa and Escherichia coli, obtained in partnership with the Special Laboratory of Clinical Microbiology/ALERTA. The enzymes obtained in this project will be used in our research group for biochemical characterization studies.