Production and purification of the rhomboid protease from the hyperthermophilic bacteria Thermotoga maritima

• Grant number: 25/21628-5
• Support Opportunities: Scholarships in Brazil - Master
• Start date: November 01, 2025
• End date: July 31, 2027
• Field of knowledge: Biological Sciences - Biochemistry - Enzymology
• Principal Investigator: Sandro Roberto Marana
• Grantee: Vitoria Hashimoto Lima
• Host Institution: Instituto de Química (IQ). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Associated research grant: 21/10577-0 - Biology of Bacteria and Bacteriophages Research Center, AP.CEPID

Abstract

Rhomboid proteases are integral membrane proteins that presents six alpha-helices, found in Bacteria, Archaea e Eukaria (Brooks, 2013). These enzymes are active upon membrane proteins, usually releasing a soluble domain with physiological function. Rhomboids activity depends on a Ser and His residues pair, placed in the helices 4 and 6, respectively. Thermotoga maritima is a hyperthermophilic anaerobic bacterium that lives in hot springs and hydrothermal vents in the ocean floor, thriving in a temperature ranging from 55 to 90°C. This bacterium produces a rhomboid protease (235 residues, 26.2 kD) presenting the usual six transmembrane helices and an additional one in its C-terminal. This project aims to produce the rhomboid protease from T. maritime as a recombinant protein in E. coli BL21(DE3), to make it soluble in micelles and then to purify this protease. Once those objectives were reached, the rhomboid protease samples could be submitted to crystallization tests and in vitro reinsertion in lipid bilayers. (AU)